A bilirubin biosensor based on a multilayer network enzyme electrode

Biosensors & Bioelectronics 10 (1995) 341-352

A bilirubin biosensor based on a multilayer network enzyme electrode Benjamin Shoham, Yoelit Migron, Azalia Riklin, Itamar Willner* & Boris Tartakovsky Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel tSavyon Diagnostics Ltd., Ashdod 77101, Israel Tel: [972] 2 666804 Fax: [972] 2 585345 (Received 23 May 1994; revised 5 August 1994; accepted 8 August 1994)

Abstract: This paper describes the construction of a bilirubin oxidase multilayer

electrode that responds amperometrically to bilirubin concentration. The enzyme electrode was constructed by the covalent attachment of bilirubin oxidase layers to a self-assembled monolayer of 3-mercaptopropionate ester, which was associated with a gold surface. Electron transfer from the enzyme redox site to the electrode was mediated by ferrocene carboxylic acid. The multilayer electrode responded linearly to bilirubin concentration in solution. In contrast to the native enzyme, which is unstable, the bilirubin oxidase multilayer electrode was stable, and its shelf-life was more than three months at 4°C. The amperometric response of the enzyme electrode towards bilirubin was dependent on the albumin concentration in the system. By adjustment of this concentration to the value present in newborns' serum, a calibration curve for the amperometric response as a function of bilirubin was deduced. The concentration of bilirubin in serum samples from newborns was determined. Keywords: Amperometric biosensor, bilirubin, enzyme monolayer, enzyme electrode, electron transfer in proteins, enzyme multilayers, bilirubine oxidase, biosensor

*To whom all correspondence should be addressed

(Waiters and G e r a r d e , 1970). This p a p e r concerns the development of a biosensor for bilirubin detection. There have been extensive attempts to develop amperometric biosensors for clinical diagnostics in recent years. A variety of redox-enzymes have been applied for the a m p e r o m e t r i c determination of a n u m b e r of biologically important analytes, such as glucose (Koudelka et al. 1991; Scheller et al. 1989), uric acid (Keedy and Vadgama, 1991), 3-hydroxybutyrate (McNeil et al., 1990), urea (Kirstein et al., 1985) and bilirubin (Wang and Ozsoz, 1990). Several methods have been used in the construction of a m p e r o m e t r i c biosen-

0956-5663/95/$07.00 © 1995 Elsevier Science Ltd

341

INTRODUCTION Serum bilirubin levels are clinically determined as part of the routine in newborn nurseries. This is because high concentrations of bilirubin (BR) in the blood m a y cause brain damage or even death, and this is especially the case in babies (Maisels, 1989). Current bilirubin analyses are based either on direct spectroscopic measurements (Westwood, 1991) or on colourimetric m e a s u r e m e n t following diazotation of the analyte

Benjamin Shoham et al.

sors, including indirect electrochemical analysis of hydrogen peroxide formed by the enzyme catalyzed oxidation of the analyte (Wang and Ozsoz, 1990), and electro-biocatalyzed oxidation of the analyte using diffusional electron mediators (Cass et al., 1984). Of particular interest are those enzyme electrodes in which the biocatalyst is immobilized onto the actual electrode surface. If such enzyme electrodes are to act as amperometric biosensors, electrical communication is required between the redox centre of the protein and the electrode. Electrical communication can be realized either by the application of a diffusional electron mediator or by the linkage of an electron transfer mediator to the enzyme electrode assembly. Covalent attachment of electron mediators to redox enzymes (Degani and Heller, 1987; Willner et al., 1992) provides a way in which electrical communication between the redox protein and the electrode surface can be established. Alternatively, immobilization of redox enzymes onto electrodes by means of redox-relay tethered copolymers (Willner et al., 1992; Gregg and Heller, 1990) has also proved effective. Recently, we developed a novel method by which enzyme-electrodes exhibiting electrical communication based on self-assembled monolayers (SAMs) could be constructed (Willner et al., 1992). In this approach, a SAM of functionalized thiols on a gold electrode acted as a base layer for the immobilization of redox proteins. Electrical communication between the enzyme redox centres and the electrode was achieved by diffusional electron mediators or covalent attachment of electron relay units to the protein in the SAM configuration. However, the protein content of the monolayer was too low to convey sufficient sensitivity for an amperometric response. Consequently, we went on to develop a method for constructing multilayer enzyme networks on the primary enzyme monolayer. This method was successfully applied in the development of a glucose biosensor based on a multilayer array of glucose oxidase assembled onto a gold electrode (Willner et al., 1993). Here we wish to report the development of a bilirubin biosensor based on an enzyme-electrode that consists of a multilayer network of bilirubin oxidase (BRO), which is built onto a thiolated, self-assembled monolayer present on the surface of a gold electrode. We demonstrate that the enzyme-electrode can be applied for amperometric determination of bilirubin. The sensitivity 342

Biosensors & Bioelectronics

of the enzyme-electrode is determined by the number of bilirubin oxidase layers associated with the electrode. Of particular interest is the observation that bilirubin oxidase, being a sensitive enzyme that is rapidly deactivated in solution (Sung et al., 1986), exhibits long-term stability in the multilayer network configuration.

EXPERIMENTAL Absorption spectra were recorded on a Kontron (Uvikon-860) spectrophotometer equipped with a thermostated cell. NMR spectra were recorded on a Bruker-WP 200 instrument. Radioactivity measurements were performed with a Beckman LS 2800 liquid scintillation counter. Electrochemical measurements were performed using a BAS CV-27 potentiostat linked to a BAS RXY recorder. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonicacid (DIDS) (disodium salt), 3,3'-dithiodipropionic acid bis(N-hydroxysuccinimide ester) (DTPS) and 6-aminohexanoic acid were purchased from Fluka. Tritiated iodoacetic acid was obtained from Du Pont. Ferrocenemonocarboxaldehyde, ferrocenemonocarboxylic acid (Fc-C), Nhydroxysuccinimide (NHS) (sodium salt), 1-ethyi3-(3-dimethylaminopropyl)carbodiimide (EDC), bilirubin lyophilized with human serum albumin (0.513/zmol of bilirubin and 1.103/xmol albumin), bilirubin oxidase (from Myrothecium verrucaria, EC 1.3.3.5, 100 units/mg) and bovine serum albumin were purchased from Sigma. All the reagents were used without further purification. Analytical grade chemicals and triple distilled water (TDW) were used to prepare all solutions.

N-(ferrocenylmethyl)-6-aminohexanoie acid (1) was prepared by the following procedure: A solution containing 0.0027 mol of ferrocenemonocarboxaldehyde (0.550 g) in 8 ml dimethylformamide was added to a solution of 6-aminohexanoic acid (containing 0.307 g, 0-0032 mol) in 2 ml of aqueous 2 ra sodium hydroxide. The mixture was heated to 80°C for 3 h. After cooling to room temperature, an excess of sodium borohydride in water was added, and the reaction mixture was stirred overnight at room temperature. After solvent evaporation, the product was recrystallized from a mixture of ethanol/diethylether. The compound gave a satisfactory elementary analysis and NMR spectrum.

Biosensors & Bioelectronics

A bilirubin biosensor based on a multilayer network enzyme electrode

The bilirubin oxidase modified by ferrocene was prepared as follows: A solution consisting of HEPES (0.100 g), urea (0.240 g), N(ferrocenylmethyl)-6-aminohexanoic acid (0-024 g), EDC (0.019 g), NHS (sodium salt) (0.0075 g) and bilirubin oxidase (0-002 g) in 2.5 ml TDW was stirred at 4°C for 20 h. The resulting mixture was dialyzed against 0.1 M HEPES buffer (pH 7.2) for 36 h. The buffer solution was subsequently changed every 12 h. The protein content was determined colourimetrically by Lowry's procedure (Lowry et al., 1951) to be 0.8 mg/ml. The amount of ferrocenyl groups bound to the protein was determined by atomic absorption. The average loading corresponded to 5 ferrocenyl groups per molecule of enzyme. Tritiated bilirubin oxidase acetic acid was prepared by the following procedure: An excess of [3H]-iodoacetic acid was added to a solution consisting of 2.06 z 10 - 7 mol bilirubin oxidase (60 units, 14 mg) in 2.5 ml 0-1 M phosphate buffer (pH 7.3). The mixture was stirred at 4°C overnight, and then purified by chromatography over Sephadex G-25. The protein concentration was determined by Lowry's procedure. The isolated fraction of labelled protein exhibited 1.6 x 104 counts.mg -1 protein. The multiple layer enzyme electrode was prepared as follows: A bare gold foil, 0.2 mm thick, with a geometrical area of ca. 0.4 cm 2, was cleaned by successive treatment with concentrated nitric acid, followed by rinsing with distilled water and dimethylsulphoxide (DMSO). The clean electrode was immersed for 2 h in a DMSO solution containing 2 x 10 2M dithiobis-(succinimidylpropionate). The monolayermodified electrode was washed twice with DMSO, and once with cold 0.1 M phosphate buffer (pH 7.3). The first bilirubin oxidase layer was covalently attached to the monolayer-modified electrode by soaking the electrode overnight at 4°C in a solution of bilirubin oxidase (100 units) in 2.5 ml phosphate buffer. A second layer of bilirubin oxidase was linked to the immobilized layer by DIDS. The enzyme electrode was dipped for 10 min in 2-5 ml cold 0-1 M phosphate buffer (pH 7.3, 0°C) containing 0.02 M DIDS. After washing the electrode twice with cold phosphate buffer, it was soaked in the enzyme solution (0°C, 30 min). This process was repeated in order to form multiple layers of bilirubin oxidase; the number of sequential modification steps

determined the number of layers attached to the enzyme network associated with the electrode. Electrochemical measurements were performed in a three-electrode cell, which contained the chemically modified electrode as working electrode, a graphite auxilliary electrode and Ag/ AgCl (KC1 3 M) reference electrode. All the potentials are reported with respect to the Ag/ AgC1 reference electrode. The working volume of the electrochemical cell was protected from light and was thermostated at 40 +- 0.5°C. The measurements were conducted using a solution of bilirubin in 0.05 M Tris buffer (pH 8.0) with 5 × 10 4 M ferrocene carboxylic acid as an electron transfer mediator. Due to the low solubility of bilirubin in water, all measurements were performed in the presence of human serum albumin, which solubilizes bilirubin. The bilirubin:albumin ratio was 0-51/~mole:l.1/~mole, unless stated otherwise. Hence, in all the experiments using bilirubin, the presence of human serum albumin should be noted. In specific experiments, the multilayer enzyme electrode was constructed with ferrocenyl-derivatized bilirubin oxidase. Analysis of blood samples was performed in a cell consisting of 1 ml of electrolyte solution. 100 ~1 serum samples were consecutively injected into the thermostated cell (40°C) and the amperometric response recorded by cyclic voltammetry (scan rate -- 2 mV-s ~).

RESULTS AND DISCUSSION Several problems are encountered during the development of a biosensor for bilirubin. Bilirubin, itself, exhibits electrochemical activity. Figure 1 shows the irreversible oxidation of bilirubin at a gold electrode. This oxidation is accompanied by the deposition of a dark coloured polymer onto the electrode, which results in an alteration of the electrode response towards bilirubin. Upon cycling of the potential, the electrode becomes insulated due to the deposition of the polymer. Thus, any biosensor for bilirubin should eliminate the direct oxidation of the analyte. The enzyme bilirubin oxidase catalyzes the oxidation of bilirubin to biliverdin by molecular oxygen, which results in the formation of hydrogen peroxide (Wang and Ozsoz, 1990) (equation 1) and water (equation 2). The fact that hydrogen peroxide is not formed stoichiometrically with respect to bilirubin (because it depends on the oxygen 343

Benjamin Shoham et al.

Biosensors & Bioelectronics

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content in the system) eliminates the possibility of basing an amperometric bilirubin sensor on hydrogen peroxide oxidation. Furthermore, bilirubin oxidase is an unstable enzyme (Sung et al., 1986) easily undergoing denaturation, and so application of this biocatalyst in a biosensor device requires its stabilization. Bilirubin + 02

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A bilirubin oxidase electrode has been assembled using the SAM approach. Firstly, a gold electrode was modified according to Scheme 1. Treatment of the gold electrode with 3,3'dithiodipropionic acid bis(N-hydroxysuccinimide ester) resulted in a SAM of the N-hydroxysuccinimide ester of 3-mercaptoproprionic acid. Subsequent modification of this functionalized monolayer with bilirubin oxidase covalently linked the enzyme to the monolayer assembly. The amount of enzyme bound to the monolayer was determined by the application of radioactively labelled bilirubin oxidase. The biocatalyst was modified with [3H]-iodoacetic acid by covalent attachment of the radioactive label to cysteine residues present on the protein (1-6-104 counts/mg protein). The radioactively labelled bilirubin oxidase was used to prepare the SAM gold electrode according to Scheme 1. The radioactive counts of the resulting bilirubin oxidase-gold monolayer electrode implied that the surface 344

coverage of the electrode by the biocatalyst corresponded to 6 × 10-12 mole/cm2 (MW of bilirubin oxidase is 68,000). The cyclic voltammogram of the bilirubin oxidase monolayer electrode in the presence of bilirubin revealed that only a background current was obtained, and that bilirubin was not oxidized directly by the electrode or by an enzyme electrocatalyzed process. Thus, attachment of bilirubin oxidase to the electrode insulated the electrode surface towards direct irreversible oxidation of bilirubin. In fact, this electrochemical insulation of the electrode by the protein is a general phenomenon. Linkage of bovine serum albumin to the active-ester of 3-mercaptopropanoate monolayer results in a similar insulation of the electrode towards direct oxidation of bilirubin (the molecular weight of bovine serum albumin, at 66,000, is very similar to that of bilirubin oxidase). Furthermore, the above experiment implies that the redox enzyme did not communicate electrically with the electrode. That is, the applied potential on the electrode did not effect the oxidation of the protein redox centre and the subsequent oxidation of bilirubin. Electrochemical oxidation of bilirubin by the bilirubin oxidase monolayer electrode was then examined in the presence of ferrocenemonocarboxylic acid (Fc-C), which acted as an electron transfer mediator. When Fc-C was present in the electrochemical cell and the bilirubin oxidase monolayer electrode was used as a working electrode, a reversible oxidation-reduction of the ferrocene compound was observed. It was interesting to note that although electrical communication between bilirubin and the electrode is blocked by the bilirubin oxidase monolayer, the electro-oxidation of Fc-C clearly occurs. This difference in behaviour can be attributed to the different electron transfer rate-constants of bilirubin and the ferrocene derivative. While the oxidation of bilirubin is slow and involves surfaceadsorbed species, the electron transfer rates of Fc-C are fast, as evidenced by the high reversibility, and hence Fc-C is able to electrically communicate with the electrode in the presence of the monolayer. Upon addition of bilirubin to the electrolyte containing Fc-C, a slight increase in the anodic current was observed. This increase in the anodic current might be attributed to the bio-electrocatalyzed oxidation of bilirubin, mediated by the FcC÷/Fc-C couple. Nonetheless, the magnitude of

Biosensors & Bioelectronics

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A bilirubin biosensor based on a multilayer network enzyme electrode

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the anodic current increase was too small to be employed as a quantitative measure of bilirubin. In fact, previous studies, which developed a glucose biosensor based on an SAM of glucose oxidase, revealed the difficulties in amperometrically monitoring glucose by a monolayer enzymeelectrode (Willner et al., 1993). The amount of enzyme immobilized in a monolayer is limited, and hence the amperometric response of the electrode is small, even when the enzyme exhibits high turnovers. To overcome these limitations, the amount of glucose oxidase on the electrode was increased by constructing an enzyme network on the base enzyme monolayer, associated with the electrode (Willner et al., 1993). Using a similar approach, we constructed a multilayer network of bilirubin oxidase on a gold electrode. Scheme 2 outlines the sequence of reactions used to modify the electrode with a bilirubin oxidase network. The enzyme monolayer-electrode was reacted with DIDS, a bifunctional reagent. This process generated an active headgroup for attachment of a second layer of enzyme. This transformation, applied in sequential additions of DIDS and bilirubin oxidase, was used to generate a tailored network of a defined number of enzyme layers. The radioactivity counts of layered bilirubin oxidase networks associated with a series of electrodes constructed by the method outlined in Scheme 2, using the [3HI-labelled enzyme, increased almost linearly with an increase in the

number of enzyme layers associated with the network (with networks comprising up to 6 layers). It should be noted that the assembled enzyme network does not necessarily reflect an organized head-to-head bilirubin oxidase linkage in consecutive layers. It is reasonable to assume that the DIDS coupling of the enzyme could have resulted in the covalent linkage of more than one enzyme molecule to a base bilirubin oxidase unit, as well as crosslinking of enzyme units within the monolayer. Nevertheless, this result indicates that, with networks comprising up to 6 layers, the enzyme content in each layer was similar to that found in the base enzyme monolayer. Thus, the organized film consisted, in fact, of bilirubin oxidase layers, bound to one another by irregular covalent linkages. Figure 2 shows the amperometric responses of a series of electrodes comprising different bilirubin oxidase layer networks, at a constant concentration of bilirubin (1.6 x 10 -4 M) and in the presence of Fc-C as an electron transfer mediator. We see that, as the number of layers in the network increases, the amperometric response becomes higher. Thus, the number of layers associated with the electrode controls the sensitivity of the resulting biosensor. It should, however, be noted that a peak current value is obtained with a network comprising 8 enzyme layers, and any further increase of the number of layers to 10 and 12 does not increase the amperometric response. Presumably, an enzyme 345

B e n j a m i n S h o h a m et al.

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Biosensors & Bioelectronics

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Biosensors & Bioelectronics

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Fig. 2. Cyclic voltammograms at 3-layer (A), 6-layer (B) and 12-layer (C) bilirubin oxidase electrodes: in the absence of bilirubin ( ), with 1.6 x lO-4 M bilirubin added (---). Electrolyte solution is composed of 0.05 M Tris buffer (pH 8) that includes 5 x 10 4 M Fc-C as electron transfer mediator. All experiments were performed at scan rate v = 2 m V • sec -1.

network of 8 layers represents an optimal configuration in which the enzyme substrate, bilirubin, can still reach the primary enzyme layers and yield the amperometric response. An increase in the number of bilirubin oxidase layers above 8 seems to insulate the base biocatalyst layers from the substrate and hence inactivate them with respect to the developed current. Therefore, subsequent studies were performed with gold electrodes comprising 8 enzyme layers in the network assembly. Control experiments revealed that when bilirubin was added to the electrochemical cell in the absence of Fc-C, the enzyme electrode (8 layers) did not develop an amperometric current. Furthermore, after deactivation of the biosensor, by heating to 95°C for 4 min, an amperometric response representing bilirubin oxidation was not obtained, in the presence of both the electron mediator and bilirubin. Similarly, an 8 layer protein assembly, comprising bovine serum albumin on a gold electrode, did not yield any anodic current in the presence of Fc-C and bilirubin that indicated the electrocatalyzed oxidation of bilirubin; only the reversible redox process of the electron mediator was observable. These results indicate that bilirubin was neither directly oxidized by bilirubin oxidase nor by the electron mediator. Figure 3 outlines the sequence of electron transfer processes resulting in the bioelectrocatalyzed oxidation of bilirubin by the bilirubin oxidase multilayer electrode and Fc-C. Fc-C is oxidized by the electrode and diffusionally mediates the oxidation of the bilirubin oxidase active site, which, in turn, oxidizes bilirubin.

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Figure 4 shows the anodic currents developed in an electrochemical cell consisting of an electrode with 8 layers of bilirubin oxidase at different concentrations of bilirubin. Figure 5 shows the steady-state currents developed in a cell consisting of the same electrode at different B R concentrations, when a constant potential, corresponding to +0.4 V, was applied to the electrode. Both the peak current vs. concentration plot, shown in Fig. 4, and the steady current vs. concentration plot, shown in Fig. 5 can be considered as calibration curves for anodic currents resulting as a function of bilirubin concentration. Another aspect requiring consideration is the stability of the multilayer enzyme electrode. Bilirubin oxidase exhibits optimal activity at 2.0

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Biosensors & Bioelectronics

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TIME(MINUTES) Fig. 5. Steady-state current developed in the electrochemical cell upon successive additions o f bilirubin from a concentrated stock solution. Each addition (indicated by solid arrow) corresponds to the generation o f a 1.7 x 10 -5 M bilirubin concentration in the electrochemical cell. Broken arrow indicates injection o f pure buffer samples o f similar volumes. The electrochemical cell consists o f the 8-layer B R O electrode, as working electrode, and the electrolyte solution is composed o f 0.05 M Tris buffer (pH 8) and 5 x 10 -4 M Fc-C. The potential applied to the working electrode was +0.4 V vs. Ag/AgCI reference electrode. The experiment was conducted at 40°C.

40°C and hence the amperometric measurements described earlier were performed at this temperature. However, bilirubin oxidase is a sensitive enzyme, and previous studies have revealed its rapid deactivation (50% activity lost within 17 h at 37°C [Sung et al., 1986]). We found that the multilayer bilirubin oxidase network electrode showed an unaltered amperometric response for a period of 8 h at 40°C. The activity of the enzyme electrode declined, however, upon longterm storage of the electrode in the electrolyte solution (40°C). The activity of the enzyme electrode was nevertheless preserved when kept in the cold (4°C) in a dry form. The amperometric responses of a series of 30 electrodes kept in the cold were examined over a period of 75 days. We observed similar anodic currents (+-5%) for the series of electrodes, which indicates that the electrodes retain their activities upon dry storage at 4°C. This result is noteworthy in view of the sensitivity of the native biocatalyst. Further experiments were carried out in an attempt to design multilayer enzyme electrodes that exclude the diffusional electron mediator. Firstly, bilirubin oxidase was chemically modified using N-(ferrocenylmethyl)-6-aminohexanoicacid (1), by covalent linkage of the electron mediator to lysine residues on the surface of the protein. 348

The Fe-content in the resulting modified enzyme was determined by atomic absorption and the protein content by Lowry's method (Lowry et al., 1951). The modified enzyme consisted of 5 moles of ferrocene per mole, and was used to construct multilayer enzyme electrodes as described in Schemes 1 and 2. The cyclic voltammograms of the electrodes consisting of 3, 6 and 9 layers of ferrocene-modified bilirubin oxidase are shown in Fig. 6. For an electrode comprising a single layer of ferrocene-modified enzyme, the charge associated with the oxidation (or reduction) of the electron mediator corresponds to 3.12/xC. Assuming that all ferrocene units linked to the bilirubin oxidase electrically communicate with the electrode, and knowing the surface coverage of the electrode from the radioactive labelling experiments (6 x 10-12 mole.cm-a), we estimated from the voltammetric responses that ca. 5 units of ferrocene were linked to each protein molecule. This result is in agreement with the loading estimated by atomic absorption. It is interesting to note that all 5 ferrocene units associated with the protein electrically communicate with the electrode surface. Presumably, one of the ferrocene units exhibits superior electron-transfer communication with the electrode, and electron exchange or hopping between this ferrocene/ferrocenylium electron-mediating site and the other ferrocene units provides the basis for effective electrical communication of the entire redox-protein. The cyclic voltammogram of the ferrocenemodified bilirubin oxidase multilayer electrode

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Biosensors & Bioelectronics

A bilirubin biosensor based on a multilayer network enzyme electrode

(8 layers) in the presence of added bilirubin shows that no electrocatalyzed anodic current was developed in the presence of the substrate. Thus, the enzyme was inactive with respect to the oxidation of bilirubin. This result might be due to the deactivation of bilirubin oxidase upon modification with the ferrocene units or construction of the multilayer, or to the lack of electrical communication between oxidized ferrocene units linked to the protein and the redox centre of the enzyme. An anodic current was, however, developed by the ferrocene-modifled bilirubin oxidase electrode (8 layers) in the presence of bilirubin and Fc-C, added to act as a diffusional electron mediator. We thus conclude that the lack of amperometric response from the ferrocene-modified enzyme electrode originated from the absence of electrical communication between the covalently linked electron mediator and the protein redox centres in the multilayer enzyme network. Modification of the protein with ferrocene units linked to the enzyme by other tether lengths or application of other relay units could facilitate an improved electrical communication in future multilayer networks. Bilirubin is almost insoluble in aqueous solution at physiological pH (pH 7.4) (Maisels, 1989) but in the blood it forms a soluble complex with albumin. Human serum albumin has a single high affinity binding site for bilirubin (Brodersen, 1982) with a stoichiometric equilibrium constant of K~ = 6 x 10 7 M - a , and one or two secondary sites that show less affinity for bilirubin, with equilibrium constants of K2 ~- K3 ~ 4"5 × 106 M-1 (Mullon et al., 1988). We might expect that the amperometric response of bilirubin, at a constant concentration, in the presence of albumin depends on the concentration of albumin added to the electrolyte solution. The amperometric response of the bilirubin oxidase multilayer electrode depended indeed, on the ratio BRt/At composing the system, where BRt represents the total bilirubin concentration and A t , the total albumin concentration. Fig. 7 shows the current resulting from a series of systems where the concentration of bilirubin in all samples was equal (0.25 mM), but the human serum albumin concentration varied (0-25, 0.315, 0.420 and 0.56 mM, or BRt/At: 1.0, 0.8, 0.6 and 0.44 respectively). It appears that the larger the concentration of total albumin, the smaller the current response. The amperometric response of the bilirubin oxidase multilayer electrode became

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negligible at high albumin concentrations. Nonetheless, the electrode retained its electrochemical activity towards the diffusional electron mediator. The decrease in the anodic current was specific towards albumin: other proteins such as papain or concanavalin A did not affect such amperometric decrease. Thus, the decrease in the amperometric response at high albumin concentrations cannot be attributed to insulation of the electrode by surface adsorbed albumin. We suggest that the bilirubin oxidase electrode responds to the concentration of free bilirubin, and hence the decrease in amperometric response with an increasing albumin concentration originates from a decline in the free bilirubin concentration. This conclusion is consistent with previous studies by Mullon et al., (1988), Wennberg et al. (1979), Brodersen and Robertson (1989) and Bramwell et al. (1990), who reported that only the free bilirubin reacts with bilirubin oxidase or peroxidase in enzymatic oxidative reactions of bilirubin in the presence of albumin. For one albumin binding site, the association of bilirubin with albumin is determined by equation 3, where Af and BRf represent free albumin and free bilirubin, respectively. The association constant of BR to A is then given by equation 4. This equation can be reformulated to give equation 5. Af + BRf = A • BR

(3)

[ A . BRI K 1 - [Af]. [BRf]

(4) 349

Benjamin Shoham et al.

Biosensors & Bioelectronics

are within _+20% of the values determined colourimetrically. It should be noted that the calibration curve relates each amperometric response to the total bilirubin in the sample (or serum). Knowing that the concentration of free bilirubin, BRf, is determined by equation 5, the concentrations of BRf in newborns can be determined at a constant albumin level.

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BRt/At BRf = K,(1 - BRt/At)

(5)

It appears from equation 5 that the BRf concentration depends on the ratio BRt/Ar, i.e. the smaller the ratio BRt/At, the smaller the BRf concentration. In order to attain amperometric responses that relate to the concentration of bilirubin in the serum of newborns, it is essential that an appropriate calibration curve is obtained (i.e. in the order of 2 to 4 mg/dl) in the presence of albumin. Figure 8 shows the experimental calibration curve of the amperometric response of the electrode towards different bilirubin concentrations at an albumin concentration that corresponds to 3 mg/dl. Table 1 summarizes the analyses of bilirubin in a series of serum samples. The bilirubin levels in these samples are determined amperometrically by the bilirubin oxidase electrode and compared to hospital results, analyzed spectroscopically. The bilirubin concentrations extracted by the amperometric method

The organization of bilirubin oxidase into layers linked to a self-assembled monolayer on a gold surface, resulted in an active enzyme electrode for the amperometric analysis of bilirubin. The bio-electrocatalyzed oxidation of bilirubin was mediated by Fc-C. The sensitivity of the enzyme electrode was controlled by the number of bilirubin oxidase layers attached to the electrode surface. The multilayer electrode eliminated direct irreversible oxidation of bilirubin. The enzyme electrode revealed a high stability when stored at 4°C. Operation of the enzyme electrode as an amperometric biosensor for bilirubin proceeded at 40°C, and under these conditions the electrode revealed reproducible analyses of bilirubin for ca. 8 h. Attempts to establish an electrical communication between the enzyme network and the electrode, by means of covalently linked ferrocenyl redox units, were unsuccessful. This might have been due to the rigidity of the layered enzyme film, or from an inadequate structure of the electron mediator. Application of ferrocenyl units with longer bridging chains, utilization of other redox mediators, such as phenothiazine, and application of other linking reagents to organize the enzyme layers, may overcome this limitation. The amperometric response of the bilirubin

TABLE 1 Bilirubin content in serum samples. Sample

1 2 3 4

350

Electrochemical analyses [BR] mg.dl-'

Colourimetric analyses [BR] mg.d1-1

13.6 6.3 20.2 6.5

11.7 7.7 16.6 8.2

Biosensors & Bioelectronics

A bilirubin biosensor based on a multilayer network enzyme electrode

oxidase electrode depended not only on the concentration of bilirubin, but also on the albumin levels in the system. The currents developed at a constant bilirubin concentration decreased as the albumin content increased. This was attributed to bilirubin binding to the albumin, and to the fact that the bilirubin oxidase electrode detects only free bilirubin. The multilayer enzyme electrode was applied as a biosensor for bilirubin in the serum of newborns. This was accomplished by extracting a calibration curve that corresponded to the amperometric response as a function of bilirubin concentration, at a level of albumin typical of that found in newborns (2-4 mg/dl). G o o d agreement in bilirubin values was found between the a m p e r o m e t r i c analyses and the currently used spectroscopic method.

ACKNOWLEDGEMENT This research was supported by Savyon Diagnostics, Ashdod, Israel.

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