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Amperometric urea biosensors based on the entrapment of urease in polypyrrole films

REACTIVE & FUNCTIONAL POLYMERS, AMSTERDAM, v. 72, n. 2, supl. 1, Part 1, pp. 148-152, FEB, 2012 http://www.producao.usp.br/handle/BDPI/37151 Downloaded from: Biblioteca Digital da Produo Intelectual - BDPI, Universidade de So Paulo

Reactive & Functional Polymers 72 (2012) 148152

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Reactive & Functional Polymers

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Amperometric urea biosensors based on the entrapment of urease in polypyrrole lms

Juliana Coatrini Soares, Andr Brisolari 1, Valquria da Cruz Rodrigues, Edgar Aparecido Sanches, Dbora Gonalves
Instituto de Fsica de So Carlos, Universidade de So Paulo, Av. Trabalhador So-carlense, 400CP 369, 13560-970 So Carlos, SP, Brazil

a r t i c l e

i n f o

a b s t r a c t
Urease (Urs) was immobilized in electrochemically prepared polypyrrole (PPy) and the resulting lms were characterized by cyclic voltammetry, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and ultraviolet visible spectroscopy (UVVIS). The enzymatic activity of Urs entrapped in the PPy matrix was conrmed by the catalytic conversion of urea into carbon dioxide and ammonia, when urea was detected amperometrically at different concentrations in standard samples and commercial fertilizers. The PPy/Urs biosensors exhibited selectivity, a relatively high efciency at urea concentrations below 3.0 mmol L1, and a sensitivity to urea of 2.41 lA cm2 mmol1 L. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 4 October 2011 Received in revised form 29 November 2011 Accepted 4 December 2011 Available online 8 December 2011 Keywords: Biosensors Conducting polymers Polypyrrole Urea Urease

1. Introduction The immobilization of enzymes in polymeric matrices is an alternative approach for preparing biosensors, which have been used to detect different types of analytes in small-volume samples. Conducting and non-conducting polymers are known as efcient host matrices for enzyme entrapment, and the performance of these biosensors depends strongly on the enzyme immobilization protocols [1,2]. Urease (Urs) has been investigated for the development of urea biosensors usually applied in environmental tests and medical diagnostics. Urs plays an important role in the determination of urea in blood, urine, wastewater, and also, during removal of urea from blood (uremia treatment) [35]. Urea is well known as the end product of protein metabolism in humans; therefore, its accumulation above critical levels should be monitored and measured periodically in serum or plasma for the detoxication of blood (hemodialysis) [6]. In addition, urea is also a soil fertilizer used as a source of nitrogen to promote the growth of many plant species. The hydrolysis of urea, which can be catalyzed by Urs, yields a typical increase in pH of the medium according to the following reaction [7,8]:
NH2 CONH2 3H2 O ! HCO 3 OH 2NH4

A suitable choice of a polymer matrix for the immobilization of Urs is polypyrrole, PPy, a highly stable, biocompatible polymer that can be prepared electrochemically at relatively low potentials in non-aqueous and aqueous media in a wide range of pH [9]. Biosensors based on the immobilization of commercially available Urs (puried) in PPy matrices have been traditionally prepared by galvanostatic [10,11] and potentiostatic [12] methods. The interactions of PPy with ammonia (gas or liquid) are also documented in the literature [1315], which offers strong evidence that reversible deprotonation of the PPy structure takes place, while a concomitant increase in the pH of the medium can be detected electrochemically. To improve the limit of detection of low-cost, accurate, and easy to use biosensors for the amperometric detection of urea, we focus here on the immobilization of Urs in PPy lms, and on the characterization of PPy/Urs lms produced electrochemically.

2. Experimental Reagent grade pyrrole (Aldrich), LiClO4 (VETEC), buffer phosphate (pH 7.0), urea (Synth), and urease (Urs) (EC 3.5.1.5, Merck, extracted from jack beans, Canavalia ensiformis) were used without further purication. All electrochemical measurements were performed with a standard three-electrode cell, with Pt, Au, or FTO (uorine-doped tin oxides) plates as working electrodes, a Pt plate, and a saturated calomel electrode (SCE) as auxiliary and reference electrodes,

Corresponding author. Tel.: +55 16 3373 9825x216; fax: +55 16 3371 5365.
1

E-mail address: gdebora@if.sc.usp.br (D. Gonalves). In memoriam.

1381-5148/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2011.12.002

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respectively. The electropolymerization of pyrrole was carried out by cyclic voltammetry in a potentiostat/galvanostat PAR Versastat II from 1.0 V to 1.0 V vs. SCE up to reach 10 complete cycles in an aqueous medium containing 0.2 mol L1 LiClO4 and 0.1 mol L1 pyrrole. This procedure allowed us to entrap Urs in the PPy lms by adding 300 lg mL1 of Urs into the electrolyte solution (0.2 mol L1 LiClO4) containing the monomer, pyrrole, at 0.1 mol L1. The value of Urs concentration, 300 lg mL1, was chosen after all of the preparation procedures of PPy/Urs lms had been optimized. Chronoamperometry was used as the transduction method for detecting urea in different solutions. The current density was measured for lms potentiostatically polarized at a xed potential of 0.28 V vs. SCE for 160 s, and in a urea concentration ranging from 1.0 to 10 mmol L1 in buffer phosphate at pH 7.0. This value of pH allowed us to achieve a condition of maximum activity of Urs. The UVVIS measurements were carried out with a Hitachi U2900 spectrophotometer for lms electrodeposited on FTO electrodes. The infrared spectra were obtained by a Thermo NicoletNexus 470/FT-IR spectrophotometer from 400 cm1 to 2000 cm1 after 32 scans for lms electrodeposited on Pt electrodes. Morphology studies were carried out with a Digital Scanning Microscope, DSM 960 (Zeiss West Germany) for lms supported on Al substrates, and then, coated with a layer of Au (about 20 nm). Commercial urea fertilizers containing different percentages of total nitrogen (45% and 75%) were comparatively used to detect their amperometric signals from the use of the PPy/Urs lms. The real samples were prepared in aqueous media containing 0.06 mg mL1 of commercial urea fertilizers (45% and 75%) in 0.2 mol L1 LiClO4. For the chronoamperometric experiments with real samples, the three-electrode electrochemical cell was also used. The solutions containing urea were introduced into the cell using a needle; after that, the current density was recorded after a short period of stabilization.

16 12 8 4

(a)
PPy/Urs

4
-2

j / mA cm

with Urs
2

PPy

0 0 2 4 6 8 10

Number of cycles

0
-2

j/ mA cm

-4

without Urs

A
4 PPy/Urs

(b)
1 0

2 PPy 0 -1

C
-2

-2

B
-3 -1.0 -0.5 0.0 0.5 1.0

E/V vs SCE
Fig. 1. Cyclic voltammograms: (a) obtained during the preparation of the PPy and PPy/Urs lms in an aqueous 0.1 mol L1 pyrrole and 0.2 mol L1 LiClO4 solution without and with 3.0 mg of Urs, respectively and (b) obtained for the PPy and PPy/ Urs lms in phosphate buffer at pH 7. The inset indicates the current density vs. number of cycles obtained at a xed potential (0.4 V vs. SCE) from the voltammograms of the preparation of the PPy and PPy/Urs lms.

3. Results and discussion Fig. 1a shows the last voltammetric curves obtained during the preparation of the PPy and PPy/Urs lms in an electrolyte solution containing pyrrole without and with Urs, respectively. The presence of Urs in the polymerization medium enables the current density to increase more rapidly and linearly with polymerization time (number of cycles). This nding is clearly illustrated in the inset of Fig. 1a for current densities recorded at a xed potential (0.4 V vs. SCE). The growth rate appears to be slower during the preparation of the PPy lm (without the enzyme), as indicated in the inset of Fig. 1a. After ten cycles, the total charge transferred during the preparation of the PPy lm was 28 mC cm2, while that of the PPy/Urs lm was 31 mC cm2, indicating that the lms growth was slightly favored in the presence of Urs. The changes in the cyclic voltammograms of the electrodes during the preparation of the lms, PPy and PPy/Urs, evidence the occurrence of electrostatic interactions between negatively charged Urs (IP 5.05.2) and its matrix, a positively charged PPy. Fig. 1b illustrates the voltammetric responses of these lms (PPy and PPy/Urs) in a monomer-free solution (phosphate buffer). The voltammetric response of the PPy lm shows the redox waves typically attributed to the PPy structure, and which have already been documented in the literature [16]. However, when Urs is entrapped in the PPy matrix, the broad processes at 0.45 V, 0.0 V, and 0.58 V vs. SCE (peaks A, B, C), typical of PPy, disappear while a new, well dened redox couple is established at 0.28 V, and 0.64 V vs. SCE (CC0 peak). Although the possible electroactive nature of Urs has been briey considered in studies on the electrochemical response of Hg electrodes with adsorbed Urs [17], it can

be assumed here that this peculiar voltammetric behavior of the PPy/Urs lm cannot be attributed to the redox response of an electroactive enzyme. In fact, it can be attributed to the nature of the polymeric matrix itself, PPy, which is a well known ion exchange and size exclusion membrane [18,19]. This nding is also consistent with previous results [11], which demonstrated that PPy/Urs lms prepared galvanostatically on Au electrodes exhibit a redox response similar to the one exhibited by our PPy/Urs lms. In our case, the voltammogram of the PPy/Urs lm shows an even better denition for the redox couple CC0 . This type of electroactive response has also been detected for PPy lms prepared in media containing large dopants with limited mobility, such as sodium dodecylbenzenesulfonate [20]. Therefore, it can be inferred that the presence of entrapped Urs may give rise to an ion exchange process during the redox scanning of the PPy/Urs lm. Instead of anions entering the PPy structure and neutralizing the charge, cations are incorporated into the PPy/Urs lm, particularly Li+ ions, thus dening the ion-exchangeable nature of the PPy matrix. The concept of this mechanism was rst described by Adeloju et al., who prepared PPy lms galvanostatically in pyrrole/NaNO3 solutions with incorporated Urs [11]. The change in the electroactive nature of the PPy lm after enzyme entrapment can be related directly to the existence of electrostatic interactions between a bulky, negatively charged enzyme entrapped in a positively charged polymer matrix, where the insertion of cations into the lm becomes well established to ensure the electroneutrality of the PPy matrix. A detailed description of the differences found in the voltammetric responses of pure PPy lms in solutions containing different anions and cations has been made previously [18]. The addition of an analyte, in this case, urea, to the buffer solution at different concentrations causes the voltammetric response of the PPy/Urs lm to change signicantly (Fig. 2a). Urea was used in a wide range of concentrations (from 1.0 102 mol L1 to

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1.0
10 10 -6 10
-5 -4

without urea
10 10
-3 -2

(a)
0.6 283

j/ A cm

-2

0.0

Absorbance/ a.u.

PPy/Urs 0.4

1,0 0,8
10
-2

buffer

j/ A cm

-1.0

-2

0,6 0,4 0,2 0,0

10 -4 10

-3

400 0.2 PPy PPy

10 -6 10

-5

-2.0 -1.0 -0.5 0.0

-0,3

0,0

0,3

0,6

0,9

E/V vs. SCE

0.0 0.5 1.0

E/V vs. ECS

400

600

800

1000

Wavelength (nm)

0.8

(b)

Fig. 3. UVVIS spectra for the PPy and PPy/Urs lms (dotted line: deconvolution curve).

0.7

0.6

0.5

0.4

peptide links (tryptophan and tyrosine residues) of Urs [25], also conrming the entrapment of Urs in the PPy matrix. The FTIR spectra of the PPy and PPy/Urs lms are quite similar, and exhibit the main bands of a predominant PPy matrix, in accordance with the literature [26,27]. For purposes of comparison, two regions in the spectra of the PPy and PPy/Urs lms are presented in Fig. 4. From 1250 cm1 to 1750 cm1, the two bands at 1460 cm1 and 1543 cm1 seen in the spectrum of the PPy lm can be

j/ A cm

-2

0.3
1385

[Urea] mol L

-1

1403

% Reflectance

1.0 106 mol L1), enabling us to obtain the current densities at the region of the peak oxidation potential (inset of Fig. 2a). The peak oxidation potential shifted continuously to more negative values after increasing the urea concentration, from 0.28 V to 0.33 V vs. SCE, therefore dening an optimal operating potential range for the activation of Urs within the PPy matrix. At a relatively low applied potential (chosen in this study as 0.28 V), one can expect minimum effects of the interference of species that are only oxidizable at higher potentials. Fig. 2b shows that the current density increases linearly in response to increasing urea concentrations at the peak oxidation potential. As expected, the pH varies only locally in response to variations in the concentration of urea, since H+ ions are consumed and ammonia is formed during the hydrolysis of urea catalyzed by Urs. Since it is well known that PPy is a pH-sensitive polymer [2123], the polymer matrix responds with a negative shift of its anodic potential when the urea concentration is increased. As related to the role of the immobilized Urs, the enzyme responds with an increase of the current density when in the presence of urea added into the solution, an effect not seen if only PPy lms are used. Another technique used here to characterize the PPy and PPy/ Urs lms was UVVIS spectroscopy (Fig. 3). The spectrum of the PPy lm shows a well-dened band at about 400 nm and a free carrier tail after about 680 nm, which are typically seen in the spectra of PPy lms with conductive properties [24]. For the PPy/Urs lm, the band at about 400 nm is visible as a shoulder in the spectrum, and a new band appears at about 283 nm due to the presence of

1294

Fig. 2. (a) Cyclic voltammograms of the PPy/Urs lm in phosphate buffer at pH 7 without and with urea at different concentrations and (b) values of current densities vs. urea concentration obtained at the peak oxidation potential.

PPy/Urs

1293

PPy
1542

1300

1400

1500

1600
-1

1700

Wavenumber (cm )

% Reflectance

665

1084

966

PPy/Urs
777

1154 968

667

773

625

PPy
913

500

600

700

800

900

1000
-1

1027

PPy

1100

1082

1152

538

1200

Wavenumber (cm )
Fig. 4. FTIR spectra of the PPy/Urs and PPy lms at two wavelength ranges.

1213

1211

PPy/Urs

1688

PPy

1458

1700

PPy/Urs

1447

1466

1621

10

-6

10

-5

10

-4

10

-3

10

-2

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attributed to the symmetric and anti-symmetric ring stretching mode of PPy, and indicate the extent of conjugation of the PPy structure [26,27]. The band at 1621 cm1 conrms the presence of Urs entrapped within the PPy matrix, since it is only visible in the spectrum of the PPy/Urs lm. Although with a low denition, the presence of this band indicates that the two spectra are in fact different, particular when one considers another band that appears at 1542 cm1, which is more intense in the spectrum of the PPy/ Urs lm. The two bands at 1621 cm1 and 1542 cm1 have generally been attributed to the C@O stretching vibration of the amide I linkage, and are typically visible in the spectrum of Urs [28] and food proteins [29], due to the presence of polypeptide bonds. The FTIR spectra of the PPy and PPy/Urs lms show practically the same bands from 500 cm1 to 1260 cm1, which are typically visible in the spectra of pure PPy. However, there are two well dened bands at 625637 cm1 which are only present in the spectrum of the PPy/Urs lms. These bands have been observed in the spectra of Urs and substituted amides, and can be assigned to a skeletal vibration of the polypeptide bonds (amide IV bands) [28,29]. In addition, the spectrum of the PPy/Urs lm in the entire spectral region shows relatively more intense bands, since several bands shared by PPy and Urs are overlapped. Fig. 5 shows the SEM micrographs of the PPy and PPy/Urs lms prepared under the same experimental conditions; the image of the PPy lm shows a cauliower-like morphology typically displayed by native PPy lms [30,31]. After Urs is immobilized, the original morphology of the lm is damaged and small spheres become visible in the micrograph of the PPy/Urs lm. These results indicate a change in the growth process of both PPy and PPy/Urs lms, as previously discussed. Fig. 6a shows the steady-state current curves for the PPy/Urs lms at different concentrations of urea and at a xed potential of 0.28 V (optimal potential). The highest values of current density were attained after a period of stabilization (shown at 200 s in Fig. 6a), and then plotted as a function of urea concentration (Fig. 6b). Adding urea to the buffer solution caused the PPy/Urs lm to become covered with ammonia produced by the Urs-catalyzed reaction. The production of ammonia continued as this reaction proceeded, causing an increase in pH and a rapid increase in the current density with urea concentrations up to 3.0 mmol L1 (see Fig. 6b). After this concentration, the current density became practically constant, and the rate of urea hydrolysis became less effective at higher concentrations (a saturation condition that occurs after about 3.0 mmol L1). The range of urea concentrations in which the analytical curve remains practically linear (from 0.5 to 3.0 mmol L1) is consistent with that of other types of urea biosensors used for the analysis of serum, urine [34], and milk [35]. The sensitivity of the PPy/Urs lm to urea was determined by the slope of the linear range of the analytical curve of Fig. 6b, showing a value of 2.4 lA cm2 mmol1 L at a maximum current density of

-20 6mM

5mM

4mM
3mM

(a)
2mM

7mM

1mM

buffer

j/ A cm-2

-40

-60

-80 0 50 100 150 200

Time (s)

-20 -21 -22

-2

(b)

j/ uA cm

-23 -24 -25 -26 -27 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

[Urea] mmol L-1

Fig. 6. (a) Chronoamperometric and (b) analytical curves obtained at different urea concentrations for the PPy/Urs lm in phosphate buffer at pH 7.0.

21.5 lA cm2, and a response time of about 3 min, which are comparable values for this type of biosensor. We also analyzed the response of the PPy/Urs lms to the concentration of urea when in medium of commercial fertilizers. Fig. 7 depicts the steady-state current curves for the PPy/Urs lm at 0.28 V vs. SCE in phosphate buffer after adding successive aliquots (10 lL of 0.1 mol L1) of fertilizer I (45% urea), fertilizer II (75% urea), urea solution (100 lL of 0.1 mol L1 urea), and phosphate buffer (100 lL of 0.1 mol L1phosphate buffer). While the curves (a) and (b) in Fig. 7 are quite similar, the curve (c) shows lower current density signals, since the concentration of urea in this solution is higher. For the last injection (curve d), the current density remains practically unchanged, indicating that the PPy/

Fig. 5. Scanning electron microscopy of the PPy and PPy/Urs lms (x10,000).

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0 -5 -10 -15
-2

(d) (a) (b) (c) Buffer

Acknowledgements The authors acknowledge nancial support of FAPESP, CAPES and CNPq. They are also grateful to Beatrice Allain for manuscript corrections and suggestions. References
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j/ A cm

-20 -25 -30 -35 -40 -45 -50 500 1000 1500 2000 Urea 45% Urea Urea 75%

Time (s)
Fig. 7. Chronoamperometric curves for the PPy/Urs lm after adding successive aliquots of fertilizer I (45% urea) (10 lL of 0.1 mol L1 fertilizer I) (a), urea (100 lL of 0.1 mol L1 urea) (b), fertilizer II (75% urea) (10 lL of 0.1 mol L1 fertilizer II) (c), and phosphate buffer (100 lL of 0.1 mol L1 phosphate buffer) (d).

Urs lm is selective only to the presence of urea solutions, particularly in commercial proportions. 4. Conclusions Amperometric biosensors for the determination of urea were successfully built by immobilizing urease (Urs) in electrochemically prepared polypyrrole (PPy) lms. The response of these biosensors to different concentrations of urea in standard solutions was linear between 0.0 and 3.0 mmol L1. Based on the chronoamperometric curves, urea was detected at a relatively high efciency up to a concentration of 3.0 mmol L1, i.e., a sensitivity of 2.4 lA cm2 mmol1 L and a response time of about 3 min. When commercial fertilizers were tested, the PPy/Urs lms were able to detect differences in the current density at concentrations of 45% and 75% of urea. Therefore, the PPy/Urs lms provide a suitable means of obtaining efcient and low-cost biosensors, since they require only the presence of pyrrole and Urs in the preparation medium and involve only a few fabrication steps.