An amperometric urea biosensor based on covalent immobilization of urease onto an electrochemically prepared copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film

An amperometric urea biosensor based on covalent immobilization of urease onto an electrochemically prepared copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film

ARTICLE IN PRESS Biomaterials 26 (2005) 3683–3690 www.elsevier.com/locate/biomaterials An amperometric urea biosensor based on covalent immobilizati...

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ARTICLE IN PRESS

Biomaterials 26 (2005) 3683–3690 www.elsevier.com/locate/biomaterials

An amperometric urea biosensor based on covalent immobilization of urease onto an electrochemically prepared copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film Rajesh, Vandana Bisht, Wataru Takashima, Keiichi Kaneto Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-Ku, Kitakyushu 808-0196, Japan Received 23 July 2004; accepted 8 September 2004

Abstract An amperometric biosensor has been developed for the quantitative determination of urea in aqueous solution. The principle is based on the use of pH-sensitive redox active dissolved hematein molecule. The enzyme, urease (Urs), was covalently immobilized on a conducting copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film, electrochemically prepared onto an indium-tin-oxide (ITO)-coated glass plate. The covalent linkage of enzyme and porous morphology of the polymer film lead to high enzyme loading and an increased lifetime stability of the enzyme electrode. Amperometric response was measured as a function of concentration of urea, at fixed bias voltage of 0.0 V vs. Ag/AgCl in a phosphate buffer (pH 7.0). The electrode gives a linear response range of 0.16–5.02 mM for urea in aqueous medium. The response time is 40 s reaching to a 95% steady-state current value, and 80% of the enzyme activity is retained for about 2 months. r 2004 Elsevier Ltd. All rights reserved. Keywords: Urea; Polymerization; Biosensor; Covalent immobilization; Peptide linkage

1. Introduction Urea is a bio-molecule that is known to play a variety of roles in the welfare of mankind. The well-known role of urea is as a fertilizer, which satisfies the nitrogen requirement of the plant. However, in human body, kidney excretes urea, an end product of protein metabolism. Blood urea nitrogen (BUN) is directly related to protein intake and nitrogen metabolism and inversely related to the rate of excretion of urea. The urea sensor becomes indispensable in diabetics monitoring to predict the nature and cause of diabetes and also as a direct indication for the onset of kidney failure or liver malfunction. The normal level of urea in serum is 8–20 mg/dl (1.3 to 3.5 mM). An increase in urea concentration causes renal failure (acute or Corresponding author. Tel.: +81 93 695 6052; +81 93 695 6052. E-mail address: [email protected] ( Rajesh).

fax:

0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.09.024

chronic), urinary tract obstruction, dehydration, shock, burns and gastrointestinal bleeding, whereas a decrease in urea concentration causes hepatic failure, nephrotic syndrome, cachexia (low-protein and high-carbohydrate diets). Many methods are available for the determination of urea, including gas chromatography, calorimetric [1,2], fluorimetric [3] analysis. However, these methods suffer from complicated sample pretreatment and are unsuitable for on-site monitoring. This inconvenience is overcome in analyzers based on electrochemical methods, in which biosensors are applied. Several types of biosensors are used for urea determination in biological samples. One is the pH-based biosensor [4–7] and the other is the NH+ 4 -based urea biosensor [8–10]. Amperometric biosensor based on enzyme, urease (Urs), is considered a promising method because of its effectiveness and simplicity. Various methods are reported for the immobilization of enzyme on different suitable matrices. Although there are large number of studies

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dealing with the development of urea biosensors [11–15] the drawbacks related to their practical use are not often underlined. In general, the main disadvantage of such biosensors is a rather narrow dynamic range for urea detection implying a necessary dilution of biological samples before measurements. Stredansky et al. [16] have recently reported the concept of an amperometric biosensor based on the use of pH-sensitive redox probe molecule, in which the enzyme Urs was physically immobilized over the water insoluble pH-sensitive redox compound, ‘lauryl gallate’ for the construction of a bulk-modified graphite composite electrode. However, the biosensor responded to a urea concentration of up to 0.75 mM in aqueous solution and showed a slow response time of 3–5 min. Recently, Urs has been immobilized on polyethylenimine film [7], for the construction of urea potentiometric biosensor but the stability of the said biosensor is only 4 weeks. Electrochemically polymerized conducting polymers had received considerable attention over the last 2 decades. The remarkable switching capability of these materials between the conducting oxidized (doped) and the insulating-reduced (undoped) state is the basis of many applications. Among others, the poly-conjugated conducting polymers were recently proposed for biosensing applications because of a number of favorable characteristics, such as (1) direct and easy deposition on sensor electrode by electrochemical oxidation of monomer, (2) control of thickness by deposition charge, and (3) redox conductivity and polyelectrolyte characteristics of the polymer useful for sensor application. Polypyrrole (PPy) fulfills the above requirements together with having the characteristics of easy oxidation, high chemical stability and low cost of monomer [17]. It was suggested [18,19] that the planar anion such as anthraquinone-2-sulfonic acid induces a charge in the PPy molecular configuration, which results in increased electrical conductivity. Because ion exchange in PPy leads to morphological changes, one can therefore utilize this process as a tool for controlling the morphology. The incorporation of a large size dopant anion, such as para-toluene sulfonate (PTS), and dodecylbenzene sulfonate (DBS) into PPy films during electropolymerization makes PPy films more porous [20]. Since porosity is an important factor for the facile immobilization of enzyme, we have immobilized enzyme on large anion-doped porous PPy film by physical adsorption technique for biosensor application to cholesterol and phenolic compounds [21–23]. Long lifetime stability of the enzyme over the matrix is an important factor in the construction of a biosensor, since this is not only beneficial to biosensor transport but also reduces per measurement cost. The major cause of poor stability is the disorption (leaching out) of enzyme from immobilization materials. As described in some studies for enzymes electrochemical entrapment

methods [24] induced by polymerization of the monomer in the presence of the bioactive moiety are simple and can be used to localize the bioactive component. However, as the biological component is randomly oriented within the polymer matrix it is often inaccessible to the target analyte [25,26]. A urea biosensor developed by Wallace et al., by simultaneous electrochemical deposition of enzyme, Urs, during polymerization of pyrrole over a platinum electrode showed decrease in background current once used for individual measurement of urea solution [27]. This degradation of the polymer was attributed to the denaturing of the enzyme. Adsorption techniques [28,29] were also used to immobilize the biological component to the outer layer of the conducting polymer to overcome the problem of burial. However, this technique suffers from the disorption of enzyme from the immobilizing material into the sample solution during measurement. Therefore, studies are required to stop the disorption of enzyme to a maximum extent from immobilization materials to obtain an increased lifetime stability of the enzyme electrode. This could be achieved only, if there would be a strong and an efficient bonding between the enzyme and the immobilizing material. As compared to other immobilization methods, carbodiimide-coupling reaction has a feature of strong covalent bonding of enzyme with the matrix, which in turn is responsible for the high enzyme loading at the surface of the electrode. In order to obtain a high enzyme loading, we propose to prepare a PTS-doped conducting copolymer having free NH2 group for covalent bonding with enzyme at its surface via carbodiimide coupling reaction. Apart from having amine functional groups, the copolymer is also having a porous morphology due to large inserted dopant para-toluene sulfonate anion. This work illustrates the versatility of an amine-based conducting poly(aminopropyl pyrrole-co-pyrrole) (PAPCP) as an immobilization platform for obtaining the high enzyme loading at the polymer surface useful for the fabrication of an enzyme electrode (Urs/PAPCP/ITO). The application of the resulting enzyme electrode for the amperometric detection of urea in aqueous medium in the presence of pH-sensitive redox active hematein molecule is described.

2. Experimental 2.1. Materials and methods Urs (EC 3.5.1.5, 102 U/mg from jack beans), 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxy-succinimide (NHS) were obtained from Sigma Chem. Co. 1-cyanoethylpyrrole monomer was obtained from Aldrich. Pyrrole monomer, p-toluene sulfonic acid sodium salt, hematein and

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urea were procured from WAKO, Japan. Pyrrole monomer was distilled thrice and the p-toluene sulfonic acid solution was freshly prepared before use. All other chemicals were of analytical grade and used without further purification.

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amine groups of the copolymer film. The copolymer electrode (PAPCP) was immersed in a phosphate buffer solution (0.1 M, pH 7.0) containing 0.015 M EDC and 0.03 M NHS for 1.5 h, and was immediately placed in an enzyme solution of 10 mg ml1 Urs in the same buffer solution for another 1.5 h. The enzyme electrode was rinsed with buffer solution (pH 7.0) to remove the excess unbound enzyme. All experiments were carried out at room temperature. The enzyme electrode was stored under dry condition at 4 1C in a refrigerator when not in use.

2.2. Preparation of monomer N-(3-aminopropyl)pyrrole 1-cyanoethyl pyrrole (0.041 mol) was added to a suspension of LiAlH4 (0.1 mol) in anhydrous ether (300 ml) and the mixture was refluxed for 20 h. After cooling, the excess hydride was destroyed by the successive addition of water (3.4 ml), a solution of 15% NaOH (3.4 ml) and water (10.2 ml). The solution was heated to 40 1C for 2 h and was filtered before evaporating to dryness. Yellow oil was obtained as a final product with a yield of about 80%. 1 H NMR d (CDCl3): 1.92 (m, 2 H, CH2-2); 2.70 (t, 2 H, CH2-3); 4.0 (t, 2 H, CH2-1); 6.15(d, 2 H, CH-b); 6.65 (d, 2 H, CH-a).

2.5. Apparatus Ultraviolet-visible (UV-vis) absorbance data were collected with a JASCO (model V-570) spectrophotometer. Fourier transform infrared (FTIR) spectra of the films were recorded on JASCO (model 230). Scanning electron micrographs were obtained with a Shimadzu; super scan (model SS-550) at an acceleration voltage of 12.0 kV. Cyclic voltammetry and amperometric measurements were done in a conventional threeelectrode cell configuration consisting of a working electrode (Urs/PAPCP/ITO), Ag/AgCl reference electrode and platinum wire as a counter electrode. A stirring bar and magnetic stirrer provided convective transport. Amperometric measurements were carried out on a cyclic voltammetry apparatus, Hokuto Denko (model HSV-100). All amperometric measurements were performed at about 25 1C in Tris-HCl buffer solution (0.001 M; pH 7.0) containing 0.1 M NaCl.

2.3. Preparation of a copolymer poly-(N-3-aminopropyl pyrrole-co-pyrrole)(PAPCP) The copolymer PAPCP film (0.25  0.25 cm2) was electrochemically prepared on an ITO glass plate from an aqueous solution containing 0.05 M pyrrole, 0.05 M (N-3-aminopropyl) pyrrole and 1.0 M p-toluene sulfonic acid sodium salt, at a fixed voltage of 0.8 V vs. SCE. The thickness of the film obtained was about 1.4 mm as calculated from the injected charge. The desired thickness was obtained at a total injected charge density of about 100 mC cm2. A total free NH2 group density of 64.8 nmol/cm2 has been found at the surface of the electrode as calculated from Kaiser test [30].

3. Results and discussion 3.1. Characterization of enzyme electrode (Urs/ PAPCP/ITO)

2.4. Fabrication of enzyme electrode (Urs/PAPCP/ ITO)

The steps involved in the fabrication of the covalent immobilization of enzyme, Urs, to a copolymer PAPCP electrode surface are shown in scheme below:

The enzyme, Urs, was covalently attached to the copolymer electrode by using the exposed surface-free

LiAlH4 Diethyl ether

N

NC

N

NH2

0.8 V vs. Ag/Ag Cl N

+

N

N

N H

H

n NH2

NH2

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Electrode

EDC + NHS

N

N

Urs

CO

H

H n

Electrode

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N

N

H

O

n

NH

NH2

C

O

Urs

The free amine groups present at the surface of the copolymer film have been utilized for the covalent attachment of enzyme, Urs, through peptide linkage with a carboxylic acid group, using the linkage reagents EDC and NHS [31,32]. The resultant enzyme electrode was characterized by FTIR spectroscopy. Fig. 1 shows FT-IR spectra of PAPCP (- - - - - -) and Urs/PAPCP (____) films. Sharp peaks were seen at 1540 cm1 and 1000–1170 cm1 in native copolymer PAPCP film, these have been assigned to CQC stretching mode and C–C stretching, respectively. However, in the spectra of enzyme immobilized polymer film (Urs/PAPCP), the peaks seen in the spectra of native film (PAPCP) at 1000–1170 cm1 have been merged into a broad peak at 950–1200 cm1 and another new sharp peak was observed at 1645 cm1. This new peak at 1645 cm1 has been attributed to CQO stretching, also called as amide I band and peak appeared at 3390 cm1 is assigned to NH deformation, also called as amide II band [33–35].

The physical morphology of the PTS–PPy film has significant role in the entrapment of the enzyme and hence reflects in the form of performance of the enzyme electrode. The morphologies of PAPCP and Urs/ PAPCP films were characterized by scanning electron microscopy (SEM). A typical SEM picture of PAPCP/ ITO (Fig. 2a) at 2.0  20000 magnification displays a three- dimensional porous open structure. This porous structure contributed a significant role towards the high enzyme stability within the polymer matrix and good reproducibility of the enzyme electrode (Urs/PAPCP/ ITO). When enzyme was immobilized in the PAPCP/ ITO matrix, many globular shape particles of 300–600 nm sizes were seen uniformly distributed through out the surface of the polymer matrix, in the SEM micrograph (Fig. 2b) at 2.5  20000 magnification. These globular-shaped particles seems to be an aggregation of protein molecules over the polymer chain. 3.2. Spectrophotometeric response studies The spectrophotometeric response of the sensor (Urs/ PAPCP/ITO) was followed by using the well-known complex formation of Nessler’s reagent (K2 Hg|| I4) with ammonia produced by the enzymatic hydrolysis of urea [36]. The key reactions are the following:

80

Urease

% Transmittance

NH2 CONH2 ðureaÞ þ H2 O ! 2NH3 þCO2 ; þ  2NH3 þ2Hgjj I2 4 ! NH2 Hg2 I3 ðlmax ¼ 385Þ þ NH4 þ5I :

60

40 PAPCP Urs/PAPCP

1000

2000

3000 -1

Wavenumber (cm ) Fig. 1. FTIR spectra of PAPCP (- - - - - - - -) and Urs/PAPCP (______) at room temperature.

Urs catalyzes the hydrolysis of urea to produce ammonia, which in turn reacts with Nessler’s reagent to form a colored product. By following the absorbance of the colored product at 385 nm, urea can be quantified and the analytical performance of the sensor was determined. We tested for the enzyme, Urs, leaching from the sensor film (Urs/PAPCP/ITO) by placing it in 2 ml TrisHCl buffer (0.001 M; pH 7.0) solution for 20 min and assay the solution for urease. The film was taken out after 20 min from the buffer solution and 1 ml of 10 mM urea solution containing 200 ml Nessler’s solution was added to check for any possible complex formation of Nessler’s reagent with ammonia produced by the

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well within the normal range of 8–20 mg dl1 urea found in human serum. 3.3. Amperometric response characteristics

(b) Fig. 2. SEM micrographs of (a) PAPCP/ITO at 2.0  20000; (b) Urs/ PAPCP/ITO at 2.5  20000.

leached out enzyme, urease, if any, on reacting with urea in the said solution. However, no detectable urease was found in the buffer solution, indicating an efficient covalent entrapment of enzyme, Urs, through peptide linkage to the exposed amino functional group at the surface of copolymer film. We have investigated the response time of the Urs/ PAPCP/ITO film to urea. The analytical response vs. time profile of our Urs/PAPCP/ITO film is shown in Fig. 3. The response is 95% of the maximum value in approximately 3 min. The response of Urs/PAPCP/ITO film to varying concentrations of urea has been delineated. The Urs/PAPCP/ITO films were placed in different urea concentrations in Tris-HCl buffer (pH 7.0) solution (2 ml) containing identical amount of Nessler’s reagent (200 ml) for 3 min before taking the absorbance at 385 nm for individual urea concentration. Fig. 4 presents a typical calibration curve for urea. The linearity for the detection of the urea concentration was found to be in the range of 0.08–5.27 mM, which falls

1.5

Absorbance

(a)

Amperometric response measurements were made with Urs/PAPCP/ITO film for the quantitative determination of urea in aqueous solution by utilizing the concept of using a water-soluble pH-sensitive dye, hematein [16]. The effect of pH on the electrochemical properties of hematein was studied. Cyclic voltammetric studies were carried out on hematein (0.5 mM) in 0.1 Mphosphate buffer at different pH values. It was seen that the cathodic peak shifted to more positive potential as pH decreased (Fig. 5). The shift in the cathodic peak potential is about 33 mV per pH unit. It is therefore expected, that the pH change be accompanied with a current change at the constant working potential. The behavior of hematein on Pt electrode was reported earlier [36]. From the practical point of view, a suitable pH-sensitive redox compound should have physical stability and should allow work at a potential near 0 mV to avoid electrochemical interference originating from real sample. Keeping this in view, a working potential of 0 mV was selected for further experiments. Fig. 6 illustrates the chronoamperometric response of the enzyme electrode at 0 mV after the addition of successive aliquots of urea to the Tris-HCl buffer solution (0.001 M; pH 7.0, 10 ml) containing 0.1 M NaCl as electrolyte and 0.5 mM hematein under a slow constant stirring of 100 rpm at 1-min time intervals, in accordance to the chronoamperometric protocol. The

1

0.5

50

100

150

200

Time (Sec) Fig. 3. Absorbance (385 nm) vs. time profile for an Urs/PAPCP/ITO film on exposure to Nessler’s reagent and 5 mM urea.

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ABSORBANCE

Absorbance

a: 1.0mM b: 1.5mM c: 3.0mM d: 4.5mM e: 6.0mM f: 7.5mM

1

0.5

2 4 6 8 Urea Concentration(mM)

1

f e d c b a 0 300

400

500

WAVELENGTH Fig. 4. Photometric calibration (l385) curve using an Urs/PAPCP/ITO film on exposure to varying concentrations of urea and identical amounts of Nessler’s reagent.

oooo

pH 10.0 pH 7.0 pH 4.0

is much shorter than those of recently reported biosensors (42 min) [15–16,37]. Fig. 7 shows the steady-state current dependence calibration curve for the each individual urea concentration. The response of enzyme electrode Urs/PAPCP/ ITO to urea was found to be linear in the range of 0.16–5.02 mM (r2=0.999). It is significant to note that this linearity range is in conformity with that obtained in the spectrophotometeric response studies. This dynamic range of urea detection is wider than recently reported biosensors, where the dynamic range is o0.75 mM [7,11–15]. The current sensitivity of the enzyme electrode obtained towards urea concentrations was 0.47 mA mM1cm2. A loss in linearity at higher urea concentration is attributed to slow surface fouling by the reaction product [22]. The lowest detection limit was calculated according to the formula 3sb/m [22,23] criteria, where m is the slope of the calibration graph and sb is the standard deviation of the blank signal. The lowest detection limit of urea electrode was found to be 0.020 mM. The reproducibility of the response of the enzyme electrode was investigated at a 5 mM urea concentration. No significant decrease in current response was observed after at least 5 uses in testing and displayed a good reproducibility. The relative standard deviation determined by 5 successive analyses of a 5 mM urea standard using a single Urs/PAPCP/ITO electrode was found to be about 12%. In a series of 10 Urs/ PAPCP/ITO electrode sensors, a relative standard deviation of about 8% was obtained for the individual current response for the same sample (5 mM urea). These

l k j i h g f e d c b a

200nA

Current( nA)

Current

2 µΑ

-0.6

-0.4

-0.2

0

0.2

0.4

a : blank b : 0.5mM c : 1.0mM d : 1.5mM e : 2.0mM f : 2.5mM g : 3.0mM h : 3.5mM i : 4.0mM j : 4.5mM K: 5.0mM l : 5.5mM

0.6

Potential (V) Fig. 5. Cyclic voltammograms of hematein on an Urs/PAPCP/ITO electrode obtained at various pHs in 0.1 M phosphate solutions; Scan rate 50 mVs1.

20

enzyme electrode responded rapidly to the urea substrate and a 95% steady-state baseline current was reached within 40 s. This response time of the electrode

40 Time (Sec)

60

Fig. 6. Typical steady-state current (chronoamperogram) response of the biosensor (Urs/PAPCP/ITO) to an increasing urea concentration.

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linkage. This efficient covalent bonding of the enzyme with amine functionalized porous PTS-doped PPy film; lead the enzyme electrode to exhibit a good performance in terms of dynamic range of detection, short response time (40 s) and long lifetime stability. The low cost and simple method of fabrication of immobilizing material is an additional advantage with respect to conventional electrodes. The experiments are presently in progress to further improve the shelf lifetime of this Urs/PAPCP/ ITO electrode beyond 2 months at 4–5 1C.

600

∆I (nA)

400

200

Acknowledgments

0

1

2

3

4

5

6

Urea concentration (mM) Fig. 7. Steady-state current dependence calibration curve of biosensor (Urs/PAPCP/ITO) to urea. Working conditions: supporting electrolyte: 0.001 M Tris-HCl buffer (pH 7.0) containing 0.1 M NaCl and 0.5 mM hematein, applied potential: 0 mV vs. Ag/AgCl.

good results may be attributed to an efficient bonding and stability of enzyme with the polymer matrix. 3.4. Sensor stability and storage conditions The Urs/PAPCP/ITO electrodes were also studied for the enzyme stability both at room temperature as well as in refrigerated conditions. The stability of the enzyme was monitored, by conducting continuous measurement of the electrode current sensitivity to a 5 mM urea sample, after an interval of 2 days for 4 months. Very slow decrement in response current was observed up to a period of 2 months. It was observed that the Urs/ PAPCP/ITO electrode retained 80% of its initial enzyme activity for 2 months, when stored at 4–6 1C in a refrigerator. This long-term stability of the Urs/PAPCP/ ITO electrode is more than the recently reported biosensors (o30 days) [7,10,14]. However, at room temperature (25 1C) the electrode lost about 70% of its initial enzyme activity within 2 weeks and thereafter showed a rapid enzyme inactivation.

4. Conclusions This study has demonstrated the feasibility of developing a conducting polypyrrole based biosensor for monitoring urea in aqueous medium. We have demonstrated that the conducting PPy having amine functional groups can be utilized as a suitable matrix for the covalent entrapment of enzyme, urease, through peptide

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