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A new amperometric biosensor for salicylate based on salicylate hydroxylase immobilized on polipyrrole film doped with hexacyanoferrate
ANALYTICA CHIMICA ACTA ELSEVIER
Analytica
Chimica
Acta 347 (1997) 35-41
A new amperometric biosensor for salicylate based on salicylate hydroxylase immobilized on polipyrrole film doped with hexacyanoferrate Benjamin G. Milagres”, Graciliano de Oliveira Netoa, Lam-o T. Kubotaa,*, Hideko Yamanakab “Institute de Quimica da Unicamp,
PO Bon 6154, 13083-970
hInstituto de Quimica-UNESP,
Received 24 September
Araraquara,
1996; received in revised form 15 January
Campinas,
SP, Brazil
SP: Brazil
1997; accepted
3 February
1997
Abstract An amperometric biosensor for salicylate detection was developed by immobilizing salicylate hydroxylase via glutaraldehyde onto a polypyrrole film doped with hexacyanoferrate, supported on a glassy carbon electrode surface. The sensor monitors the catechol produced in the enzymatic reaction on the film surface, at an applied potential of 150 mV vs. SCE. A [NADH]/[salicylate] ratio between 2 and 4 gave the best response. The biosensor presented the best performance in a solution with pH=7.4. The response time was about 40 s. A linear range of response was observed for salicytate concentrations between 1.0x lo-’ and 1.0x 10e4 mol 1-l and the equation adjusted for this curve was 1=(-0.04~0.01)+(11.4rt0.2)[sa1icy1ate] with a correlation coefficient of 0.999 for n=6. The biosensor retains its activity for at least 10 days despite daily use. The results obtained using the biosensor for salicylate determination, in three different samples of antithermic drugs, presented a good correlation with the standard calorimetric method. Keywords:
Salicylate
biosensor;
Polypyrrole
film; Salicylate
hydroxylase;
1. Introduction Nowadays
salicylate
in its derivatized
forms
is
[1,2]. Salicylate is the main metabolite resulting from hydrolysis of acetyl salicylic acid and its amount in the blood and urine of patients who are therapeutically treated with medicine containing salicylate derivatives [3] must be determined as a higher level than 2.2 mol 1-l of salicylate is toxic for the patients [4]. The determination of saliwidely
used in medicine
*Corresponding 0003-2670/97/$I?.oO PII
author. Fax: +55 019 239 3805
0
1997 Elsevier Science
SOOO3-2670(97)00105-O
B.V. All rights reserved.
Amperometry
cylates in dermatological preparations, pharmaceuticals and foods, where it has long been used for preservation [5], is also very important. A large number of methods for salicylate determination has been described in the literature [6-81. Although widely used, the spectrophotometric methods suffer interference from several compounds 19, lo]. Spectrofluorimetric methods [ 11,121 give better results but require clean samples. Liquid or gas chromatographic methods have been used simultaneously to determine several derivatives of salicylic acid [13,14]. Liquid membrane electrodes [ 15,161, allowing for fast determinations, have been
36
B.G. Milagres et al. /Analytica
developed, however, their selectivity and stability are poor. The high selectivity and specificity of enzymes are very attractive to solve interference problems [ 171. However, the enzymes are expensive and may increase the cost of the analysis. Thus, immobilization of the enzyme for repeated use makes the analysis less expensive. The use of salicylate hydroxylase (E.C. 1.14.13. l), which converts salicylate, in presence of NADH and oxygen, to catechol, NAD+ and carbon dioxide, has been employed for selective determination of salicylate [ 18,191. The reaction between salicylate and salicylate hydroxylase occurs according to the following equation [20]: Enzyme
Salicylate +NAD+
+ NADH + 2H+ + 02
F!
Catechol
+ 2H20 + COZ
The consumption of NADH [21] or oxygen [22] or the formation of catechol [23] or carbon dioxide [24] can be monitored and are proportional to the salicylate concentration. An amperometric biosensor constructed with the enzyme salicylate hydroxylase attached directly onto the platinum working electrode together with quinone was recently described in literature [23], however, the applied potential is 450 mV vs. SCE. The potential used in this case is high and presents some interference problems. The use of a mediator in the biosensor has been extensively used to reduce the potential to about -100 to 0 mV vs. SCE [25] in ideal conditions to avoid interferences such as ascorbic acid, urate, cysteine, etc. [26-281. In a previous work, we have reported a biosensor, made with carbon paste modified with silica gel, that senses catechol at 200 mV vs. SCE [29]. However, the carbon paste electrode is not very clean to use in clinical analysis. Polypyrrole doped film may electrocatalyze the catechol oxidation [30]. This work describes the preparation of electropolymerized polypyrrole doped with hexacyanoferrate to reduce the necessary potential to oxidise catechol. The immobilization of the salicylate hydroxylase via glutaraldehyde and development of a biosensor for salicylate determination using low applied potential are also presented.
Chimica Acta 347 (1997) 35-41
2. Experimental 2.1. Pyrrole electropolymerization A glassy carbon disk electrode (Methrom) of 3 mm diameter, polished with alumina (0.3 and 0.1 pm) and electrochemically cleaned cycling 10 times in a solution of 3 mol 1-l HzS04 was used to electropolymerize the polypyrrole film. The electropolymerization was carried out with an electrochemical cell containing 5 ml of an aqueous solution of 0.1 mol I-’ of pyrrole freshly distilled (Aldrich) and potassium ferrocyanide (Riedel) at same concentration. All experiments were carried out using a PAR-273A potentiostat connected to a microcomputer, a Saturated Calomel Electrode (SCE) as reference electrode and a platinum wire as counter electrode. The solution was deoxygenated for 10 min by purging with nitrogen before begin the electropolymerization. The applied potential was 0.7 V vs. SCE and electropolymerization time was changed from 50 to 150 s. After electropolymerization the polypyrrole film was washed with demineralized water and one cycle of cyclic voltammetry, scanned at 20 mV SK’ between -0.2 and 0.5 V vs. SCE, was carried out to complete the electropolymerization. 2.2. Enzyme immobilization An alliquot of 25 ul of a 5% (w/v> glutaraldehyde(Fluka) solution containing 30 U of salicylate hydroxylase (E.C. 1.14.13.1 from Sigma) and 1 pg of albumin (Aldrich) was put onto the electropolymerized polypyrrole film on the glassy carbon electrode surface and dried at room temperature. The dilution of glutaraldehyde was carried out using a 0.1 mol 1-l phosphate buffer solution (pH=7.4). 2.3. Sample preparation Samples of three different brands (Melhoral”, AAS@ and Aspirin@) were prepared by trituration with a mortar and pestle. This triturated material was added to 10.00 ml of a 0.5 mol 1-i NaOH solution and this mixture was refluxed for 1 h. The resulting mixture was filtered and neutralized with 0.5 mol ll’ sulfuric acid solution and quantitatively transferred to a 50.00 ml volumetric flask. The volume was
B. G. Milagres et al. /Analytica
completed with demineralized water. The necessary dilutions were carried out using a 0.1 mol I-’ phosphate buffer solution (pH=7.4).
37
Chimica Acta 347 (1997) 35-41
_:' .:'
200 -
:'
,:' :
100
3. Results and discussion ~o~___-.;I
3.1. Preparation
of polypyrrole
film -100 -
The time of electropolymerization showed a significant inthtence on the film properties. When the electropolymerization time was 150 s the film was apparently more resistive and less adherent, however no significant increase in incorporated HCF was observed. When the electropolymerization time was 100 s, a similar resistivity to that obtained when the time was 50 s was observed, but the adherence was not as good, although a slight increase in the amount of incorporated HCF was observed when 100 s was used for electropolymerization. Based on the good quality, such as adherence, mechanical resistance, resistivity and quantity of incorporated mediator observed in the film electroplymerized for 50 s, this time was chosen to construct the biosensor. The obtained film showed a dark color (black), smooth and shining. The incorporation of ferrocyanide or other ions was verified by cyclic voltammetry. 3.2. Electrochemical
properties
Fig. 1 presents the cyclic voltammograms obtained with (A) glassy carbon, (B) glassy carbon with electropolymerized polypyrrole film doped with chloride (PPyCl) and (C) glassy carbon with film doped with ferrocyanide (PPyHCF). The voltammograms (A) and (B) are very similar, indicating the absence of electroactive species in the film. A slight increase in capacitive current in voltammogram (B) is observed, suggesting that a small increase of the electrochemical active surface occurred. However, when ferrocyanide is incorporated in the polypyrrole film a redox couple peak and a large increase in the capacitive current is observed. The increase of capacitive current was assigned to the increase of the electrode surface indicating that the film is polymerized in a different way, presumably the film growth leaves the “cavities” or in a fiber form [31] that allows diffusion of the electrolyte. These differences between the films elec-
j
-200 -
;......
-300
1
:. -0.1
,
I 0.0
.
. . .‘.
B
A
.:’
: _’
:
:
:
I
I
0.1
0.2
E/V
I 03
I
I 04
vs SCE
Fig. 1. Cyclic voltammogramsobtained with glassy carbon (A), glassy carbon modified with PPyICI (B) and glassy carbon modified with PPylHCF (C) electrodes, in 0.1 mol I-’ phosphate buffer solution (pH 7.4) and a scan rate of 20 mV s- ‘.
tropolymerized with chloride or ferrocyanide is attributed to differences in the electropolymerization mechanism. The redox couple peaks observed in the voltammogram with a midpoint potential of 0.10 V vs. SCE was attributed to the Fe(II)/Fe(III) couple of the hexacyanoferrate. This midpoint potential is lower than that observed in neutral solution. Thus, this decrease in the midpoint potential may be assigned to stabilization of the oxidized form of the HCF due to basicity of the polymer (electron donor character). The linear correlation between peak current and scan rate observed by the plot (Fig. 2) indicates that the electroactive species are strongly adhered onto the electrode surface. Cyclic voltammograms obtained in different supporting electrolytes showed that the cations such as sodium, lithium, potassium and ammonium do not affect the electrochemical response showing the same AE, value i.e. 95 mV. In contrast, larger cations such as dimethyl-ammonium provokes an increase in AE, (AE,=356 mV), suggesting that they diffuse through the film. The mobility of the ions through the film is very important to provide electrochemical behavior, so the hydrated ionic radius of the electrolyte should be lower than that of the cavity of the polymeric film.
38
B.G. Milagres et al. /Analytica
Chimica Acta 347 (1997) 3541
-400 -
I
-600
-1.5
-1 .o
,
I
I
-0.5
0.0
0.5
EI’J
’
-3ooL -0.4
’ -0.2
’
’ 0.0
’
’
-
0.2
’
’
0.4
’
1
0.6
I B
EIV vs SCE
vs SCE
Fig. 2. Cyclic voltammograms obtained with glassy carbon modified with PPyMCF electrode at different scan rates: 5 (A), 10 (B), 20 (C), 30 (D), 40 (E) and 50 (F); in 0.1 mol 1-r phosphate buffer solution (pH 7.4). Inset, a graph showing anodic peak current dependence on scan rate.
Fig. 3. modified solution (B) after (C) after
Table 1 Cyclic voltammetry parameters different solution pH values
shown in Fig. 3(B). On the other hand, the film presented a good electrocatalytic property to oxidize catechol, as can be seen in Fig. 3(C). In this way, the sensor could be used to construct a biosensor for salicylate, monitoring the catechol formed during the enzymatic reaction. Fig. 4 shows the ampero-
for the biosensor
PH
W&c
Era (mV)
&
6.8 7.4 8.1
0.89 0.95 1.00
178 176 174
20 30 80
(mV)
obtained
F,
at
(mV)
99 103 127
An interesting observed effect of the pH on electrochemical response of the film is presented in Table 1. The ratio Z,,/Z,, tends to unity, when the pH of the solution is increased. The midpoint potential also increases with the pH, in contrast to the behavior generally observed in electrochemical processes [32], but AE, decreases. A most interesting fact is the increase of the cathodic peak potential while the anodic peak potential remains almost constant. This behavior indicates that the electrochemical process of the mediator incorporated into the film has become more reversible [33], when the solution pH is increased. 3.3. Electrocatalytic
properties
The electropolymerized PPy film doped with HCF presented no electrocatalytic property for NADH, as
Cyclic voltammograms obtained with glassy carbon with PPyMCF electrode in a 0.1 mol 1-l phosphate (pH 7.4). at scan rate of 20 mV SK’: (A) with no addition. addition of 50 ul of a 0.10 mol 1-l NADH solution and addition of 50 ul of a 0.1 mol I-’ catechol solution.
0-
6-
_
a
I
i 4A
B
22 0.9
\ 1.0
J 1.1
Time I ks Fig. 4. The current obtained in a chronoamperometry experiment, with an applied potential of 150 mV vs. SCE, after addition of: (A) 50 ul of a 0.1 mall-’ NADH solution and (B) 50 u1 of a 0.1 mol 1-l catechol solution, in 5 ml of 0.1 mall-’ phosphate buffer (pH 7.4).
E.G. Milagres et al. /Analytica
39
Chimica Acta 347 (1997) 3541
metric response for NADH and catechol at potential of 150 mV vs. SCE. As can be observed, the current for catechol at same concentration of NADH is very high. The current for NADH is negligible compared with the current for catechol. When the film was used without glutaraldehyde, the mediator is leached out of the polypyrrole film, indicating that the glutaraldehyde form a film to avoid the leaching of HCF. 3.4. Biosensor
response
n
1%
-
.- -.--~-.
1.
/
1.
‘.O- ./
IO
-
1
O.O!
,
,
1
2
I
,
3
I
I
I
4
5
r
1
6
[NADH] / [Salicylatel
Fig. 5. Effect of [NADH]/[salicylate] ratio on biosensor response. Applied potential of 150 mV vs. SCE in 0.1 mol 1-l phosphate buffer (pH 7.4).
30
40
50
60
I pm01 L-’
Fig. 6. Calibration curves for the salicylate biosensor at different solution pH: 6.1 (A), 7.4 (B) and 8.1 (C). Applied potential of 150 mV vs. SCE.
0.6 i
20
[salicylate]
0.8 -
f 2
, : _.i:’
p:/&f
The modified electrode was used to sense catechol formed during the enzymatic reaction. As mentioned earlier, the NADH is not oxidized at low applied potential. The addition order of NADH and salicylate was tested and no influence on the response was observed, suggesting that the electrode responds only to catechol. Adding NADH first and then salicylate, the response was 1.94 (1tO.09) pA and when salicylate was added first, the current was 1.95 (~tO.09) PA, for a salicylate concentration of 1.0x lop4 mol l- ’ at an applied potential of 150 mV vs. SCE. The NADH quantity influenced the biosensor response as shown in Fig. 5. An excess of NADH could improve the response [23], however, for a [NADH]/[salicylate] ratio higher than 4, a decrease of the response was observed. This behavior may be
J
attributed to an excess of NADH that impedes salicylate diffusion to the film interface. A reasonable range of 2 to 4 for the [NADH]/[salicylate] ratio was verified to give the best responses of the biosensor. Thus, this ratio should be carefully considered during salicylate determination in real samples. The response time, i.e. the time necessary for the signal to reach the maximum after substrate addition, for the biosensor was about 40 s. This time is shorter than that observed for other salicylate biosensors [23]. This behavior may be very important for the speed of the analysis for use of this biosensor in a flow injection system to implement in clinical laboratories for routine analysis. Calibration curves at different solution pH are shown in Fig. 6. A higher sensitivity is observed for the curve obtained in the solution with pH 7.4, which is similar to that soluble enzyme 1201. A calibration curve obtained for a freshly prepared biosensor, applying 150 mV of potential, showed a linear range of response for salicylate concentration from 1.0x 1O--5 to 1.0~10-~~ mol I ‘. keeping the [NADH]/[salicylate] ratio between 2 and 4. The equation of the curve was: I=(-0.04&0.01)+( I 1.4f 0.2)[salicylate] with a correlation coefficient of 0.999 for n=6. The precision of the measurements was assessed by conducting triplicate measurements at [salicylate]=S.Ox 1O-5 mol 1-l. The mean catalytic
40
B.C. Milagres et al. /Analytica
Table 2 Amount of acetyl salicylic acid found in antithermic standard method and using the biosensor Sample
Amount found per tablet (mg)” Standard
Aspirin@ AAS@ Melhora@
drugs by a
method
503.615.4 500.0& 6.7 495.4f7.1
Biosensor
Nominal
502.3f7.0 499.4f4.1 489.8f8.2
500.0 500.0 500.0
current was found to be 1.97 pA with a standard deviation of 0.03 PA, indicating a great precision. The stability of the biosensor was tested by making a calibration curve daily and the slope changed from 11.4(f0.2) to 7.6(f0.3) after 10 days. This decrease can be attributed to the denaturation of some of the immobilized enzyme molecules. When not in use the biosensor was kept in a phosphate buffer pH 7.4 in a refrigerator at 5°C. determination
5.0x lop6 mol 1-l is lesser than 2 order of magnitude warrants a great sensibility for salicylate detection. The response time, stability and repeatability are better than the other reported in literature. However the pH and [NADH]/[salicylate] ratio should be carefully controlled.
Acknowledgements
a Deviation for three determinations.
3.5. Salicylate
Chimicu Acta 347 (1997) 35-41
in real samples
The biosensor performance was verified by determining salicylate in three different drugs (Aspirin@, AAS@ and Melhoral@). The drugs present a nominal quantity of acetyl salicylic acid of 500.0 mg per tablet. The tablets were submitted to hydrolysis, as previously described [34]. These hydrolyzed samples were analyzed in triplicate with the biosensor and using a standard calorimetric method from Sigma’s kit. According to the results listed in Table 2, a good correlation between the methods is verified.
4. Conclusions A film obtained here presented a good adherence and mechanical resistance allowing the use the biosensor during 10 days, or about 100 analysis. The film formed by the glutaraldehyde to immobilize the enzyme also avoids the leaching of the mediator from the film, increasing the stability of the electrode. A good electrocatalytic efficiency is observed in electroxidation of the catechol at a low applied potential compared with those found in the literature, and it may be attributed to the HCF that should electrocatalyze the oxidation of catechol. The detection limit of
The nomic for the Collins
authors thank FAPESP and European EcoCommunity (contract no. CIl*-CT93-0029) financial support. They also thank Prof. Carol for English revision of the manuscript.
References [I] J.E. Frew, SW. Bayliff, P.N.B. Gibbs and M.J. Green, Anal. Chim. Acta, 273 (1989) 39. [2] K.D. Rainsdorf, Aspirin and the Salicylates, Butterworths, London, UK, 1984, pp 56-58 and 245-248. [3] B.E. Chem, D. Johns, F. Bochner, O.M. Imhoff and M. Howland, Clin. Chem., 25 (1979) 1420. [4] T.C. Kwong, Clin. Chem. News, 1 (1985) 14. [51 ES. Kang, T.A. Todd, M.T. Capaci, K. Schwenzer and J.T. Jabbour, Clin. Chem., 29 (1983) 1012. [61 M.J. Stewart and I.D. Watson, Ann. Clin. Biochem., 24 (1987) 552. 171 G.A. Rivas and J.M. Calatayud. Talanta, 42 (1995) 1285. PI P.B. Bertocchi, D. D’Ottavio, M.E. Evangelist, M. Mascini and G. Palleschi, Clin. Chim. Acta, 207 (1992) 205. [91 K.S. You and J.A. Bitticofer, Clin. Chem., 30 (1984) 1549. [lOI U. Saha and K. Baksi, Analyst, 110 (1985) 739. [Ill J.A.M. Pulgarin and A.A. Molina, Analyst, 119 (1994) 1915. [W A. Villari, N. Micali, M. Frest and G. Puglisi, Analyst, 119 (1994) 1561. 10 [I31 M. Carlson and R.D. Thompson, J. Liquid Chromathogr., (1987) 997. [I41 J.N. Buskin, R.A. Upton and R.L. Willians, Clin. Chem., 28 (1982) 1200. [I51 A.M. Papaglou, E.P. Diamandis and T.P. Hadjiioannou, Anal. Chim. Acta, 159 (1984) 393. [161 S.M. Hassan and M.A. Hamada, Analyst, 113 (1988) 1709. [I71 M.A.T. Gilmartin and J.P. Hart, Anal. Proc., 32 (1995) 341. [181 T.J. Moore, M.J. Joseph, B.W. Allen and L.A. Coury, Anal. Chem., 67 (1995) 1896. [I91 M. Neumayr, 0. Friedrich and G. Sontag, Anal. Chim. Acta, 273 (1993) 469. WI M.A.N. Rahni, G.G. Guilbault and G. de Oliveira Neto, Anal. Chim. Acta, 181 (1986) 219. WI L. Gorton, G. Bremle, E. Csoregi, G.J. Pettersson and B. Persson, Anal. Chim. Acta, 249 (1991) 43.
B.C.
1221
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et ah/Anal~tica
KS. You, Clin. Chim. Acta, 149 (1985) 281. 231 M. Neumayr, Ci. Sontag and F. Pittner, Anal. Chim. Acta, 305 (1995) 26. .24] T. Fonong and G.A. Rechnitz, Anal. Chim. Acta. 158 (1984) 357. .25] L. Gorton, Electroanalysis, 7 (1995) 23. :26] L. Gorton, H.J. Karan, P.D. Hale, T. Inagaki, Y. Okamoto and T.A. Skothein, Anal. Chim. Acta, 228 (1990) 23. [27] J. Wang, L.H. Wu, Z. Lu, R. Li and J. Sanches, Anal. Chim. Acta, 228 ( 1990) 25 I. 1281 C.G. Borges, G. de Oliveira Neto, L.T. Kubota and L. Grandin, J. Electroanal. Chem., 418 (1996) 147.
Chimica
Acta
347
(1997)
3541
II
[29] L.T. Kubota, B.G. Milagres, F. Gouveia and G. de Oliveira Neto, Anal. Lett., 29 (1996) 893. 1301 S. Dong and J. Ding, Synth. Metals, 24 (1988) 273. [31] L.S. Van Dyke and C.R. Martin, Synth. Metals, 36 (1990) 275. 1321 A.B.P. Lever, Inorg. Chem.. 29 (1990) 127 I. [33] R.W. Murray, Chemically Modified Electrodes. in: A.J. Bard (Ed.), Electroanalytical Chemistry. vol. 13. Marcel Dekker Inc., NY, 1984, pp. 191-368. [34] K.K. Choi and K.W. Fug, Anal. Chim. Acta, I38 (1982) 385.
Analytica
Chimica
Acta 347 (1997) 35-41
A new amperometric biosensor for salicylate based on salicylate hydroxylase immobilized on polipyrrole film doped with hexacyanoferrate Benjamin G. Milagres”, Graciliano de Oliveira Netoa, Lam-o T. Kubotaa,*, Hideko Yamanakab “Institute de Quimica da Unicamp,
PO Bon 6154, 13083-970
hInstituto de Quimica-UNESP,
Received 24 September
Araraquara,
1996; received in revised form 15 January
Campinas,
SP, Brazil
SP: Brazil
1997; accepted
3 February
1997
Abstract An amperometric biosensor for salicylate detection was developed by immobilizing salicylate hydroxylase via glutaraldehyde onto a polypyrrole film doped with hexacyanoferrate, supported on a glassy carbon electrode surface. The sensor monitors the catechol produced in the enzymatic reaction on the film surface, at an applied potential of 150 mV vs. SCE. A [NADH]/[salicylate] ratio between 2 and 4 gave the best response. The biosensor presented the best performance in a solution with pH=7.4. The response time was about 40 s. A linear range of response was observed for salicytate concentrations between 1.0x lo-’ and 1.0x 10e4 mol 1-l and the equation adjusted for this curve was 1=(-0.04~0.01)+(11.4rt0.2)[sa1icy1ate] with a correlation coefficient of 0.999 for n=6. The biosensor retains its activity for at least 10 days despite daily use. The results obtained using the biosensor for salicylate determination, in three different samples of antithermic drugs, presented a good correlation with the standard calorimetric method. Keywords:
Salicylate
biosensor;
Polypyrrole
film; Salicylate
hydroxylase;
1. Introduction Nowadays
salicylate
in its derivatized
forms
is
[1,2]. Salicylate is the main metabolite resulting from hydrolysis of acetyl salicylic acid and its amount in the blood and urine of patients who are therapeutically treated with medicine containing salicylate derivatives [3] must be determined as a higher level than 2.2 mol 1-l of salicylate is toxic for the patients [4]. The determination of saliwidely
used in medicine
*Corresponding 0003-2670/97/$I?.oO PII
author. Fax: +55 019 239 3805
0
1997 Elsevier Science
SOOO3-2670(97)00105-O
B.V. All rights reserved.
Amperometry
cylates in dermatological preparations, pharmaceuticals and foods, where it has long been used for preservation [5], is also very important. A large number of methods for salicylate determination has been described in the literature [6-81. Although widely used, the spectrophotometric methods suffer interference from several compounds 19, lo]. Spectrofluorimetric methods [ 11,121 give better results but require clean samples. Liquid or gas chromatographic methods have been used simultaneously to determine several derivatives of salicylic acid [13,14]. Liquid membrane electrodes [ 15,161, allowing for fast determinations, have been
36
B.G. Milagres et al. /Analytica
developed, however, their selectivity and stability are poor. The high selectivity and specificity of enzymes are very attractive to solve interference problems [ 171. However, the enzymes are expensive and may increase the cost of the analysis. Thus, immobilization of the enzyme for repeated use makes the analysis less expensive. The use of salicylate hydroxylase (E.C. 1.14.13. l), which converts salicylate, in presence of NADH and oxygen, to catechol, NAD+ and carbon dioxide, has been employed for selective determination of salicylate [ 18,191. The reaction between salicylate and salicylate hydroxylase occurs according to the following equation [20]: Enzyme
Salicylate +NAD+
+ NADH + 2H+ + 02
F!
Catechol
+ 2H20 + COZ
The consumption of NADH [21] or oxygen [22] or the formation of catechol [23] or carbon dioxide [24] can be monitored and are proportional to the salicylate concentration. An amperometric biosensor constructed with the enzyme salicylate hydroxylase attached directly onto the platinum working electrode together with quinone was recently described in literature [23], however, the applied potential is 450 mV vs. SCE. The potential used in this case is high and presents some interference problems. The use of a mediator in the biosensor has been extensively used to reduce the potential to about -100 to 0 mV vs. SCE [25] in ideal conditions to avoid interferences such as ascorbic acid, urate, cysteine, etc. [26-281. In a previous work, we have reported a biosensor, made with carbon paste modified with silica gel, that senses catechol at 200 mV vs. SCE [29]. However, the carbon paste electrode is not very clean to use in clinical analysis. Polypyrrole doped film may electrocatalyze the catechol oxidation [30]. This work describes the preparation of electropolymerized polypyrrole doped with hexacyanoferrate to reduce the necessary potential to oxidise catechol. The immobilization of the salicylate hydroxylase via glutaraldehyde and development of a biosensor for salicylate determination using low applied potential are also presented.
Chimica Acta 347 (1997) 35-41
2. Experimental 2.1. Pyrrole electropolymerization A glassy carbon disk electrode (Methrom) of 3 mm diameter, polished with alumina (0.3 and 0.1 pm) and electrochemically cleaned cycling 10 times in a solution of 3 mol 1-l HzS04 was used to electropolymerize the polypyrrole film. The electropolymerization was carried out with an electrochemical cell containing 5 ml of an aqueous solution of 0.1 mol I-’ of pyrrole freshly distilled (Aldrich) and potassium ferrocyanide (Riedel) at same concentration. All experiments were carried out using a PAR-273A potentiostat connected to a microcomputer, a Saturated Calomel Electrode (SCE) as reference electrode and a platinum wire as counter electrode. The solution was deoxygenated for 10 min by purging with nitrogen before begin the electropolymerization. The applied potential was 0.7 V vs. SCE and electropolymerization time was changed from 50 to 150 s. After electropolymerization the polypyrrole film was washed with demineralized water and one cycle of cyclic voltammetry, scanned at 20 mV SK’ between -0.2 and 0.5 V vs. SCE, was carried out to complete the electropolymerization. 2.2. Enzyme immobilization An alliquot of 25 ul of a 5% (w/v> glutaraldehyde(Fluka) solution containing 30 U of salicylate hydroxylase (E.C. 1.14.13.1 from Sigma) and 1 pg of albumin (Aldrich) was put onto the electropolymerized polypyrrole film on the glassy carbon electrode surface and dried at room temperature. The dilution of glutaraldehyde was carried out using a 0.1 mol 1-l phosphate buffer solution (pH=7.4). 2.3. Sample preparation Samples of three different brands (Melhoral”, AAS@ and Aspirin@) were prepared by trituration with a mortar and pestle. This triturated material was added to 10.00 ml of a 0.5 mol 1-i NaOH solution and this mixture was refluxed for 1 h. The resulting mixture was filtered and neutralized with 0.5 mol ll’ sulfuric acid solution and quantitatively transferred to a 50.00 ml volumetric flask. The volume was
B. G. Milagres et al. /Analytica
completed with demineralized water. The necessary dilutions were carried out using a 0.1 mol I-’ phosphate buffer solution (pH=7.4).
37
Chimica Acta 347 (1997) 35-41
_:' .:'
200 -
:'
,:' :
100
3. Results and discussion ~o~___-.;I
3.1. Preparation
of polypyrrole
film -100 -
The time of electropolymerization showed a significant inthtence on the film properties. When the electropolymerization time was 150 s the film was apparently more resistive and less adherent, however no significant increase in incorporated HCF was observed. When the electropolymerization time was 100 s, a similar resistivity to that obtained when the time was 50 s was observed, but the adherence was not as good, although a slight increase in the amount of incorporated HCF was observed when 100 s was used for electropolymerization. Based on the good quality, such as adherence, mechanical resistance, resistivity and quantity of incorporated mediator observed in the film electroplymerized for 50 s, this time was chosen to construct the biosensor. The obtained film showed a dark color (black), smooth and shining. The incorporation of ferrocyanide or other ions was verified by cyclic voltammetry. 3.2. Electrochemical
properties
Fig. 1 presents the cyclic voltammograms obtained with (A) glassy carbon, (B) glassy carbon with electropolymerized polypyrrole film doped with chloride (PPyCl) and (C) glassy carbon with film doped with ferrocyanide (PPyHCF). The voltammograms (A) and (B) are very similar, indicating the absence of electroactive species in the film. A slight increase in capacitive current in voltammogram (B) is observed, suggesting that a small increase of the electrochemical active surface occurred. However, when ferrocyanide is incorporated in the polypyrrole film a redox couple peak and a large increase in the capacitive current is observed. The increase of capacitive current was assigned to the increase of the electrode surface indicating that the film is polymerized in a different way, presumably the film growth leaves the “cavities” or in a fiber form [31] that allows diffusion of the electrolyte. These differences between the films elec-
j
-200 -
;......
-300
1
:. -0.1
,
I 0.0
.
. . .‘.
B
A
.:’
: _’
:
:
:
I
I
0.1
0.2
E/V
I 03
I
I 04
vs SCE
Fig. 1. Cyclic voltammogramsobtained with glassy carbon (A), glassy carbon modified with PPyICI (B) and glassy carbon modified with PPylHCF (C) electrodes, in 0.1 mol I-’ phosphate buffer solution (pH 7.4) and a scan rate of 20 mV s- ‘.
tropolymerized with chloride or ferrocyanide is attributed to differences in the electropolymerization mechanism. The redox couple peaks observed in the voltammogram with a midpoint potential of 0.10 V vs. SCE was attributed to the Fe(II)/Fe(III) couple of the hexacyanoferrate. This midpoint potential is lower than that observed in neutral solution. Thus, this decrease in the midpoint potential may be assigned to stabilization of the oxidized form of the HCF due to basicity of the polymer (electron donor character). The linear correlation between peak current and scan rate observed by the plot (Fig. 2) indicates that the electroactive species are strongly adhered onto the electrode surface. Cyclic voltammograms obtained in different supporting electrolytes showed that the cations such as sodium, lithium, potassium and ammonium do not affect the electrochemical response showing the same AE, value i.e. 95 mV. In contrast, larger cations such as dimethyl-ammonium provokes an increase in AE, (AE,=356 mV), suggesting that they diffuse through the film. The mobility of the ions through the film is very important to provide electrochemical behavior, so the hydrated ionic radius of the electrolyte should be lower than that of the cavity of the polymeric film.
38
B.G. Milagres et al. /Analytica
Chimica Acta 347 (1997) 3541
-400 -
I
-600
-1.5
-1 .o
,
I
I
-0.5
0.0
0.5
EI’J
’
-3ooL -0.4
’ -0.2
’
’ 0.0
’
’
-
0.2
’
’
0.4
’
1
0.6
I B
EIV vs SCE
vs SCE
Fig. 2. Cyclic voltammograms obtained with glassy carbon modified with PPyMCF electrode at different scan rates: 5 (A), 10 (B), 20 (C), 30 (D), 40 (E) and 50 (F); in 0.1 mol 1-r phosphate buffer solution (pH 7.4). Inset, a graph showing anodic peak current dependence on scan rate.
Fig. 3. modified solution (B) after (C) after
Table 1 Cyclic voltammetry parameters different solution pH values
shown in Fig. 3(B). On the other hand, the film presented a good electrocatalytic property to oxidize catechol, as can be seen in Fig. 3(C). In this way, the sensor could be used to construct a biosensor for salicylate, monitoring the catechol formed during the enzymatic reaction. Fig. 4 shows the ampero-
for the biosensor
PH
W&c
Era (mV)
&
6.8 7.4 8.1
0.89 0.95 1.00
178 176 174
20 30 80
(mV)
obtained
F,
at
(mV)
99 103 127
An interesting observed effect of the pH on electrochemical response of the film is presented in Table 1. The ratio Z,,/Z,, tends to unity, when the pH of the solution is increased. The midpoint potential also increases with the pH, in contrast to the behavior generally observed in electrochemical processes [32], but AE, decreases. A most interesting fact is the increase of the cathodic peak potential while the anodic peak potential remains almost constant. This behavior indicates that the electrochemical process of the mediator incorporated into the film has become more reversible [33], when the solution pH is increased. 3.3. Electrocatalytic
properties
The electropolymerized PPy film doped with HCF presented no electrocatalytic property for NADH, as
Cyclic voltammograms obtained with glassy carbon with PPyMCF electrode in a 0.1 mol 1-l phosphate (pH 7.4). at scan rate of 20 mV SK’: (A) with no addition. addition of 50 ul of a 0.10 mol 1-l NADH solution and addition of 50 ul of a 0.1 mol I-’ catechol solution.
0-
6-
_
a
I
i 4A
B
22 0.9
\ 1.0
J 1.1
Time I ks Fig. 4. The current obtained in a chronoamperometry experiment, with an applied potential of 150 mV vs. SCE, after addition of: (A) 50 ul of a 0.1 mall-’ NADH solution and (B) 50 u1 of a 0.1 mol 1-l catechol solution, in 5 ml of 0.1 mall-’ phosphate buffer (pH 7.4).
E.G. Milagres et al. /Analytica
39
Chimica Acta 347 (1997) 3541
metric response for NADH and catechol at potential of 150 mV vs. SCE. As can be observed, the current for catechol at same concentration of NADH is very high. The current for NADH is negligible compared with the current for catechol. When the film was used without glutaraldehyde, the mediator is leached out of the polypyrrole film, indicating that the glutaraldehyde form a film to avoid the leaching of HCF. 3.4. Biosensor
response
n
1%
-
.- -.--~-.
1.
/
1.
‘.O- ./
IO
-
1
O.O!
,
,
1
2
I
,
3
I
I
I
4
5
r
1
6
[NADH] / [Salicylatel
Fig. 5. Effect of [NADH]/[salicylate] ratio on biosensor response. Applied potential of 150 mV vs. SCE in 0.1 mol 1-l phosphate buffer (pH 7.4).
30
40
50
60
I pm01 L-’
Fig. 6. Calibration curves for the salicylate biosensor at different solution pH: 6.1 (A), 7.4 (B) and 8.1 (C). Applied potential of 150 mV vs. SCE.
0.6 i
20
[salicylate]
0.8 -
f 2
, : _.i:’
p:/&f
The modified electrode was used to sense catechol formed during the enzymatic reaction. As mentioned earlier, the NADH is not oxidized at low applied potential. The addition order of NADH and salicylate was tested and no influence on the response was observed, suggesting that the electrode responds only to catechol. Adding NADH first and then salicylate, the response was 1.94 (1tO.09) pA and when salicylate was added first, the current was 1.95 (~tO.09) PA, for a salicylate concentration of 1.0x lop4 mol l- ’ at an applied potential of 150 mV vs. SCE. The NADH quantity influenced the biosensor response as shown in Fig. 5. An excess of NADH could improve the response [23], however, for a [NADH]/[salicylate] ratio higher than 4, a decrease of the response was observed. This behavior may be
J
attributed to an excess of NADH that impedes salicylate diffusion to the film interface. A reasonable range of 2 to 4 for the [NADH]/[salicylate] ratio was verified to give the best responses of the biosensor. Thus, this ratio should be carefully considered during salicylate determination in real samples. The response time, i.e. the time necessary for the signal to reach the maximum after substrate addition, for the biosensor was about 40 s. This time is shorter than that observed for other salicylate biosensors [23]. This behavior may be very important for the speed of the analysis for use of this biosensor in a flow injection system to implement in clinical laboratories for routine analysis. Calibration curves at different solution pH are shown in Fig. 6. A higher sensitivity is observed for the curve obtained in the solution with pH 7.4, which is similar to that soluble enzyme 1201. A calibration curve obtained for a freshly prepared biosensor, applying 150 mV of potential, showed a linear range of response for salicylate concentration from 1.0x 1O--5 to 1.0~10-~~ mol I ‘. keeping the [NADH]/[salicylate] ratio between 2 and 4. The equation of the curve was: I=(-0.04&0.01)+( I 1.4f 0.2)[salicylate] with a correlation coefficient of 0.999 for n=6. The precision of the measurements was assessed by conducting triplicate measurements at [salicylate]=S.Ox 1O-5 mol 1-l. The mean catalytic
40
B.C. Milagres et al. /Analytica
Table 2 Amount of acetyl salicylic acid found in antithermic standard method and using the biosensor Sample
Amount found per tablet (mg)” Standard
Aspirin@ AAS@ Melhora@
drugs by a
method
503.615.4 500.0& 6.7 495.4f7.1
Biosensor
Nominal
502.3f7.0 499.4f4.1 489.8f8.2
500.0 500.0 500.0
current was found to be 1.97 pA with a standard deviation of 0.03 PA, indicating a great precision. The stability of the biosensor was tested by making a calibration curve daily and the slope changed from 11.4(f0.2) to 7.6(f0.3) after 10 days. This decrease can be attributed to the denaturation of some of the immobilized enzyme molecules. When not in use the biosensor was kept in a phosphate buffer pH 7.4 in a refrigerator at 5°C. determination
5.0x lop6 mol 1-l is lesser than 2 order of magnitude warrants a great sensibility for salicylate detection. The response time, stability and repeatability are better than the other reported in literature. However the pH and [NADH]/[salicylate] ratio should be carefully controlled.
Acknowledgements
a Deviation for three determinations.
3.5. Salicylate
Chimicu Acta 347 (1997) 35-41
in real samples
The biosensor performance was verified by determining salicylate in three different drugs (Aspirin@, AAS@ and Melhoral@). The drugs present a nominal quantity of acetyl salicylic acid of 500.0 mg per tablet. The tablets were submitted to hydrolysis, as previously described [34]. These hydrolyzed samples were analyzed in triplicate with the biosensor and using a standard calorimetric method from Sigma’s kit. According to the results listed in Table 2, a good correlation between the methods is verified.
4. Conclusions A film obtained here presented a good adherence and mechanical resistance allowing the use the biosensor during 10 days, or about 100 analysis. The film formed by the glutaraldehyde to immobilize the enzyme also avoids the leaching of the mediator from the film, increasing the stability of the electrode. A good electrocatalytic efficiency is observed in electroxidation of the catechol at a low applied potential compared with those found in the literature, and it may be attributed to the HCF that should electrocatalyze the oxidation of catechol. The detection limit of
The nomic for the Collins
authors thank FAPESP and European EcoCommunity (contract no. CIl*-CT93-0029) financial support. They also thank Prof. Carol for English revision of the manuscript.
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