Urea potentiometric biosensor based on urease immobilized on chitosan membranes

Urea potentiometric biosensor based on urease immobilized on chitosan membranes

Talanta 47 (1998) 183 – 191 Urea potentiometric biosensor based on urease immobilized on chitosan membranes Ju´lia M.C.S. Magalha˜es, Ade´lio A.S.C. ...

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Talanta 47 (1998) 183 – 191

Urea potentiometric biosensor based on urease immobilized on chitosan membranes Ju´lia M.C.S. Magalha˜es, Ade´lio A.S.C. Machado * LAQUIPAI, Faculdade de Cieˆncias, R. Campo Alegre 687, Porto P4150, Portugal Received 12 September 1997; received in revised form 5 February 1998; accepted 11 February 1998

Abstract Potentiometric biosensors based on urease (E.C. 3.5.1.5.) immobilized on chitosan membranes coupled to all-solid-state nonactin ammonium ion selective electrodes are described. The enzyme was immobilized on the chitosan membranes by four procedures: (A) adsorption; (B) adsorption followed by reticulation with dilute aqueous glutaraldehyde solution; (C) activation with glutaraldehyde followed by contact with the enzyme solution; and (D) activation with glutaraldehyde, contact with the enzyme solution and reduction of the Schiff base with sodium borohydride. The response characteristics of the biosensors obtained with these enzymatic membranes were determined and compared. The biosensor with best response characteristics, obtained by procedure (B), showed the following characteristics of response to urea: (i) linearity in the 10 − 4 to 10 − 2 M range; (ii) slope of up to 56 mV per decade; (iii) response time between 30 s and 2 min; and (iv) lifetime of 2 months. This biosensor was tested in the determination of urea in blood serum samples. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Urea potentiometric biosensor; Urease; Chitosan membrane; Blood serum

1. Introduction Urease is an important enzyme in biological systems, where it catalyses the conversion of urea to carbon dioxide and ammonia. The coupling of this catalytic reaction with different tranducers has allowed the development of thermal [1–6], amperometric [7 – 10], conductimetric [11 –14], optical [15–18], piezoelectric [19], potentiometric [20–44], and FET based [45,46] biosensors. Such biosensors have been receiving attention since * Corresponding author. Tel.: + 351 2 6082874; fax: + 351 2 6082959; e-mail: [email protected]

their introduction in 1969 [20,21], but recently a upsurge of interest can be detected in the literature (almost 25% of the papers listed under References were published in or after 1995). Potentiometric biosensors, based on the detection of either ammonium ion [20–31], ammonia gas [32–37], carbon dioxide [38] or pH change [39–41] produced by the enzymatic reaction, are among the most attractive biosensors for urea, because of the simplicity of their construction procedure and the general availability of the instrumentation required for their utilization. Commercial self-contained electrode probes based on ammonia gas selective electrodes coupled to im-

0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0039-9140(98)00066-6

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mobilized urease are available [47]. Previous work [29,30] showed that all-solid-state nonactin based ammonium ion selective electrodes are among the most adequate transducers for the development of potentiometric urea biosensors, due to their fast and reproducible response [26], and their long lifetime, up to 12 months [27]. The development of such potentiometric biosensors for determination of urea in blood serum deserves much interest, since urea is a biological product that is monitored in blood as an indicator of renal function. Enzyme immobilization plays a fundamental role in the performance characteristics of biosensors based on ion selective electrodes. Ideally, a large amount of the active enzyme should be directly attached to the surface of the electrode membrane in a matrix without a diffusional barrier. However, the development of a biosensor for blood serum analysis not only requires that the sensing membrane polymer has adequate functional moieties for stable enzyme immobilization, but also that this polymeric material shows good compatibility with blood serum. PVC membranes do not fulfil these two conditions but sensing membranes made of polymers with better compatibility with blood proteins, like silicone [48], or with membranes of modified polymers to allow better enzyme immobilization, for instance modified PVC [49], usually show a less selective response than PVC based electrodes. Moreover membranes have been coupled to ion selective electrodes to modify the selectivity of their response, in which the pore size molecular cut-off of dialysis membranes [26] or transient electrostatic effects, for example in anion exchange membranes [28], are explored. The immobilization of urease on a membrane matrix with good blood compatibility and with good permeability to ammonium is desirable for the development of potentiometric enzyme biosensors based on ammonium selective electrodes. Chitin, a linear polymer composed of near straight chains of b-(1 “ 4)2-acetamido-2-deoxyD-glucopyranose, kept together by strong interchain hydrogen bonding, is the second most abundant natural polysaccharide after cellulose.

Upon deacetylation of chitin a related substance, chitosan, is obtained [50]. Both substances have been used for enzyme immobilization for industrial applications [51] and, as they are non-toxic and biocompatible [52], they are suitable as support materials for the construction of biosensors for measurements in blood serum samples. Very recently, enzymatic sensors with chitin and chitosan supports have been reported for determination of glucose [53], lactate [54], ethanol [55] and urea [56]. The literature on immobilization of urease on chitosan is dominated by the use of beads [57], but Krajewska et al. [58] immobilized urease on membranes of chitosan activated with glutaraldehyde. However, the chemistry of chitosan/glutaraldehyde reactions is complex [59] and the permeability of the membranes to several ions, including ammonium, decreases when the ratio of glutaraldehyde/chitosan used in the preparation is increased [60]. On the other hand, when the immobilization is based on Schiff base formation, leaching of the enzyme from the membrane can occur due to the reversibility of this reaction [61]. The aim of the present work was to develop a potentiometric urea biosensor, based on urease immobilized on a chitosan membrane, applied to an all-solid-state ammonium electrode. To obtain membranes with high enzyme loadings, with adequate permeability towards ammonium ion and without significant enzyme leaching, four different procedures of enzyme immobilization were attempted: (A) adsorption; (B) adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions; (C) activation with glutaraldehyde followed by contact with the enzyme solution; and (D) activation with glutaraldehyde, contact with the enzyme solution and reduction of the Schiff base with sodium borohydride. The characteristics of the biosensors assembled with these membranes were studied and compared. A preliminary report of this work [56] included only results obtained by procedure (A). Biosensors with type B membranes showed the best response characteristics and were tested for the determination of urea in real samples of blood sera.

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2. Experimental

2.1. Preparation of chitosan membranes A solution was prepared by overnight stirring of 1 g of chitosan (Sigma) in 100 ml of 0.8% (w/v) acetic acid. The membranes were cast on polyethylene plates on a nylon mesh from a measured volume per surface area of 0.34 ml/cm2, and formed upon drying at 60°C overnight. The next day they were neutralized with 1% NaOH solution for 30 min and washed with water. The membranes were kept under water until use for enzyme immobilization.

2.2. Enzyme immobilization The procedures described below were used.

2.2.1. Adsorption based procedures 2.2.1.1. Procedure A (physical adsorption) [62]. The membranes were dipped in a pH 4 acetic acid solution, washed with water and then left overnight at ca. 5°C, in contact with an urease solution containing 2 mg of the enzyme (urease III, Sigma) per ml of a pH 5.6 phosphate buffer. The next day, the membranes were washed with water and with a pH 7.0 phosphate buffer solution. 2.2.1.2. Procedure B (adsorption followed by reticulation) [63]. Type A membranes were reticulated with a 0.01% glutaraldehyde solution for 60 min and then washed with water. All the membranes, types A and B, were kept in a pH 7.0 phosphate buffer until use. 2.2.2. Acti6ation based procedures 2.2.2.1. Procedure C (acti6ation with glutaraldehyde) [64]. One of the sides of a membrane (2 cm2) was activated with 0.02 ml of a 1% glutaraldehyde solution and was allowed to dry. Then, 0.02 ml of an urease solution with 2 mg/ml in a pH 5.6 phosphate buffer was spread on the same surface and left until dry. The membrane was washed with water and kept in a pH 7.0 phosphate buffer until use.

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2.2.2.2. Procedure D (acti6ation followed by reduction with sodium borohydride) [65]. Membranes of type C were treated with a sodium borohydride solution (5 mg/ml) dissolved in a pH 9.0 borate buffer. The reduction with this solution was carried out at ca. 5°C for 10 min. The membrane was washed with water and kept in a pH 7.0 phosphate buffer until use. 2.3. Determination of the acti6ity of the immobilized enzyme Measurements were made with an ammonium selective electrode (see Section 2.4) following a procedure described by Mascini and Palleschi [37]. Fifty milliliters of 0.1 M urea solution in 0.1 M (pH= 7) TRIS buffer were placed in a cell and, after stabilization of the potential difference, a piece of membrane with area previously measured (2–4 cm2) was introduced into the solution and the variation of potential with time was recorded. The amount of ammonium ion produced along the time was calculated from a previously obtained calibration curve and the specific immobilized activity per unit area was calculated in mmol min − 1 cm − 2 (unit cm − 2)

2.4. Construction of the ammonium electrode All-solid-state ammonium ion selective electrodes with a nonactin cocktail dispersed in a PVC membrane applied on a conductive epoxy support [66] were used as base electrodes. Graphite powder (B50 mm, Merck) was used to make the epoxy (Epoxy Technology, H54-UNF) conductive. The membranes were constituted by evaporation of the solvent from a THF (Merck p.a.) solution of nonactin (Fluka Selectophore, Ionophore I) (2%), bis(2-ethylhexyl)adipate (Fluka, Selectophore) (68%) and PVC (Fluka, selectophore) (30%).

2.5. Assembly of the biosensors The membranes with immobilized urease were applied to the tip of the ammonium electrodes (F=10 mm) with a silicone ring.

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2.6. E6aluation of the biosensors

3. Results and discussion

Calibration curves in the range 0.02 –20 mM were obtained by titration of 20 ml of a 0.1 M (pH= 7) TRIS buffer with a 0.1-M urea solution prepared with the same buffer as solvent.

3.1. Effect of the chitosan membranes on the ammonium electrode response

2.7. Other equipment The electrodes were calibrated at 259 0.2°C and an Orion 90.02 double junction reference electrode (with TRIS buffer in the external compartment) was used. The calibrations were carried out with an automatic system controlled by a Compaq personal computer, Prolinea 325S. The potential difference values were acquired with the AD converter of a Lab Master DMA (Scientific Solutions) card, through a high impedance circuit. A Crison Microbu2030 microburete, controlled via the RS 232C interface of the computer, was used for the addition of the standard solution.

Similarly to cellulosic membranes, chitosan membranes are water swollen gels that can affect the response characteristics of the enzymatic electrodes. Calibration curves of the ammonium electrode showed that the slope of its response to ammonium was slightly lowered (Table 1) when the electrode was assembled with chitosan membranes without immobilized enzyme and that the lower limit of linear response was shifted from 5× 10 − 5 to 1× 10 − 4 M. Chitosan membranes with urease immobilized by adsorption (procedure A) showed no influence on the response to ammonium ion, but for electrodes assembled with membranes reticulated with glutaraldehyde (procedure B), the response was affected in about the same way as for the chitosan membrane (Table 1).

2.8. Blood serum analysis For the determinations, a urea biosensor and an ammonium electrode were used simultaneously and a differential measurement was made both in calibration and sample measurement [67]. A solution [26] containing ammonium chloride (0.0003 M), potassium chloride (0.002 M) and sodium chloride (0.140 M) was diluted tenfold with 0.1 M (pH= 7.0) TRIS buffer and used as a matrix for the calibration of the urea biosensor in the range 0.0001–0.02 M. Blood serum samples were also diluted (1:10) with 0.1 M (pH=7.0) Tris buffer. The blood serum samples were kindly provided by the Laborato´rio de Ana´lises Clı´nicas of the Faculty of Pharmacy of Oporto. The results provided by this laboratory were used for comparison. They were obtained with an automatic analyzer using a two step enzymatic method based on urea to ammonium transformation by urease, followed by reaction of this ion with a-ketoglutarate in the presence L-glutamic dehydrogenase and NAD (nicotinamide adenine dinucleotide).

Table 1 Effect of the chitosan membranes on the response of the ammonium selective electrodea Membrane

R

s

Eo

Without chitosan membrane

0.99996

57.9 9 0.4

212.6 9 0.9

0.99991 0.99997

57.4 9 0.3 57.9 9 0.4

208.6 90.8 213.6 91.0

Chitosan

0.9998 0.9998 0.9995

51.9 90.5 52.7 90.4 52.3 90.3

197.8 91.2 197.3 91.0 196.5 90.8

A

0.9998 0.9997 0.9996

58.4 90.5 58.3 90.5 57.8 90.7

213.0 91.4 212.9 91.4 210.4 91.6

B

0.9999 0.9999 0.9992

51.2 90.9 55.0 90.4 56.6 9 2.0

192.1 90.9 201.2 9 0.9 202.7 9 5.2

a Parameters obtained in the concentration range 5×10−4 to 10−2 M. R, correlation coefficient (n = 6); s, slope in mV/ decade; E o in mV, with reference to an Orion 90-02 reference electrode, both given with standard deviation.

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during a couple of months. Biosensors of type B showed lifetimes longer than 2 months, while those of type A showed lifetimes of 1 month. Biosensors of type B showed higher slopes of response to urea than those of type A. The stability along time of the response potential of these biosensors to urea solutions (Table 2 and Fig. 2) showed that the response of biosensors of type B was more stable. The differences observed in the

Fig. 1. Calibration curves of (urea) biosensors of types B and C.

3.2. Response characteristics of the urea biosensors The immobilization of urease on chitosan by physical adsorption (procedure A) produced membranes with high activity of immobilized enzyme (19 units cm − 2). Reticulation of these membranes with glutaraldehyde (procedure B) decreased slightly the enzyme activity to 17 units cm − 2 (with SD = 91.5, n =3). Both activation based procedures rendered membranes with much lower activity (1 – 2 units cm − 2 with SD= 90.3, n=3). Despite of this large difference in enzymatic activity, all the membranes produced urea biosensors with potentiometric response. Biosensors of types A and B showed linear response (response potential versus logarithm of urea concentration) in the range 1 ×10 − 4 to 1 ×10 − 2 M, while biosensors of types C and D showed linear response in the range 5× 10 − 4 to 1×10 − 2 M (Fig. 1). This difference in the range of linear response is related to the permeability characteristics of the membranes [68] and the large difference in their enzymatic activity [69]. Besides the different values for the lower limit of linear response, Fig. 1 also shows a marked difference in the shape of the calibration curves obtained for biosensors of types B and C, which is a consequence of the large difference in enzymatic activity of the two types of membranes [69]. Table 2 summarizes the calibration parameters of the urea biosensors obtained along the time,

Fig. 2. Variation along the time of the response to a 2 × 10 − 3 M urea solution of biosensors of the four types.

Eo 167.09 2.2 192.19 3.3 112.79 2.2 107.19 2.4 89.69 3.2 90.29 2.2 39.19 3.5 71.39 3.5 52.39 5.1

s 43.9 9 0.9 47.4 91.3 36.6 9 0.9 42.4 9 0.9 39.5 91.3 46.5 9 0.8 41.4 9 1.5 47.6 91.4 34.5 9 2.0

See Table 1 footnote.

0.9992 0.9991 0.9997 0.9992 0.9988 0.9998 0.9996 0.9990 0.9990

1 2 7 8 11 15 24 27 40

a

R

Days

Biosensor A

1 2 3 4 7 11 24 40 63

Days 0.9997 0.9995 0.9997 0.9993 0.9997 0.9992 0.9993 0.9995 0.9999

R

Biosensor B

52.39 0.4 45.8 9 1.2 52.2 9 1.2 56.6 9 0.7 43.3 90.6 40.8 9 0.7 48.1 9 0.8 43.1 9 0.7 46.9 9 0.7

s 142.891.3 145.79 2.7 156.49 3.2 140.99 1.7 128.59 0.9 101.19 1.6 101.79 2.6 147.59 1.9 161.89 1.7

Eo

Table 2 Calibration parameters of the urea biosensors A – D obtained on different daysa

1 3 4 7 8 9 17 20

Days 0.9996 0.9983 0.9999 0.9999 0.9980 0.9973 0.9999 0.9900

R

Biosensor C

47.4 91.5 31.6 9 1.2 46.9 92.0 54.2 9 1.6 58.3 92.2 31. 8 9 1.5 36.3 92.2 29.2 9 1.3

s

71.4 9 4.0 29.0 9 3.2 60.9 9 5.0 5.9 9 4.3 81.5 9 5.8 34.09 4.0 96.7 9 5.7 −15.19 3.4

Eo

1 3 10 13 21 24 38

Days

0.9997 0.9997 0.9997 0.9990 0.9996 0.9998 0.9997

R

Biosensor D

52.3 9 1.4 37.8 91.6 37.3 9 2.3 35.8 91.2 38.0 91.4 39.1 90.9 32.0 92.4

s

142.893.5 91.14.394.1 15.713.095.9 106.29 3.0 99.393.7 110.79 2.3 20.79 6.2

Eo

188 J.M.C.S. Magalha˜es, A.A.S.C. Machado / Talanta 47 (1998) 183–191

J.M.C.S. Magalha˜es, A.A.S.C. Machado / Talanta 47 (1998) 183–191

patterns of variation in Fig. 2, which shows a more marked decrease for type A than for type B biosensors, are related to the reticulation step in the procedure of enzyme immobilization. Biosensors of types A and B showed good response reproducibility within an working day, with standard deviations of the slope and E o obtained in three consecutive calibrations lower than 1.5 mV/ decade and 4 mV, respectively. Biosensors of type C had a lifetime of only 2 weeks and showed a noisy response as shown by the values obtained for the parameters of the calibration curves (Table 2) and by the marked oscillations of the potentials in response to a 2× 10 − 3 M urea solution obtained on different days (Fig. 2). The results obtained with biosensors of type D showed that reduction of the Schiff base with sodium borohydride produced no improvement of the response characteristics. Indeed biosensors of type D showed much lower slopes than those of type C except on the first day (Table 2). The response time of the present urea biosensors varied from 30 s to 3 min depending on the urea concentration and on the type of the biosensor. The shortest response times were obtained for biosensors of type A (30 s to 2 min) and the longest for biosensors of type C (2 – 3 min). The response times for type B biosensors varied between 30 s and 2 min. These results show that the best procedure for urease immobilization on chitosan membranes for obtaining potentiometric urea biosensors is adsorption of the enzyme followed by reticulation with dilute aqueous glutaraldehyde solutions. The urea biosensors assembled with these membranes (type B), showed characteristics competitive with those of other potentiometric urea biosensors based on coupling immobilized urease to ammonium selective electrodes [20 – 27]. Type B biosensors show longer lifetimes than biosensors based on urease immobilized in polyacrilamide [23,24]. The results also show that reticulation of the adsorbed enzyme significantly improves its stability as shown by the reproducibility of response and lifetime of type B biosensors. The linear response range and response time of type B biosensors are identical to most potentiometric

189

Table 3 Results obtained in the determination of urea in samples of blood sera Sample

1 2 3 4 5 6 7 8 9 10 11 12 a b

Urea (mg/100 ml) Provideda

Foundb

39 62 46 19 23 38 28 32 63 27 62 45

38.2 9 0.9 64.0 9 2.0 44.0 9 1.0 23.0 9 0.4 24.9 9 0.2 37.0 9 1.0 31.2 9 0.6 33.2 9 0.7 62.0 92.0 29.0 90.4 63.0 91.0 46.9 90.8

See text. Average of three determinations with standard deviation.

biosensors based on coupling of urease to ammonium ion selective electrodes based on nonactin [23–27].

3.3. Analysis of blood sera Table 3 summarizes the results obtained in the determination of urea in samples of blood sera using a biosensor of type B. The value of the correlation coefficient (R) for the least squares linear regression of the urea concentration values determined with the potentiometric biosensor versus the values provided by the clinical analysis laboratory was 0.994, showing a good correlation between the results obtained by the two methods. The slope of the regression line is rather low, 0.94, but falls within the confidence limits (95% level) of 0.87 and 1.02. The value of the intercept for the regression line is 3.3 with confidence limits (95% level) of 0.15 and 6.49. This suggests that the method used for matrix correction, based on the results obtained in simultaneous measurements of ammonium with a ion selective electrode and urea with the biosensor of type B, is not fully adequate for the purpose of matrix correction.

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4. Conclusions Chitosan is an adequate material for the preparation of potentiometric urea biosensors based on coupling of enzymatic membranes with all-solid state PVC membrane ammonium electrodes with nonactin as sensor. The characteristics of response of these urea biosensors depend on the procedure used for enzyme immobilization. The biosensors with urease immobilized on chitosan membranes by adsorption followed by crosslinking with dilute aqueous glutaraldehyde solutions, showed the best characteristics of response, including the longest lifetime (more than 2 months).

Acknowledgements Dra. Laura Pereira of the Laborato´rio de Ana´lises Clı´nicas of the Faculty of Pharmacy of Oporto is thanked for providing serum samples and results of spectrophotometric measurements of their urea contents.

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