229

Enzyme Electrode for the Determination of Bilirubin Aluin Fortuney' and George G. Guilhault *+,'-' + Department of Chemistry, University of New Orleans, Lakefront, LA 70148. USA

Present address: Department of Chemistry, University College of Cork, Cork, Ireland ++

Received: March 23, 1995 Final version: May 12, 1995

Abstract Bilirubin oxidase (EC 1.3.3.5) catalyzes the oxidation of bilirubin to biliverdin with the concurrent reduction of oxygen to hydrogen peroxide. A platinum electrode was prepared with an immobilized bilirubin oxidase membrane. Bilirubin was determined by monitoring the increase in hydrogen peroxide concentration from the enzyme reaction. The calibration plot for bilirubin shows linearity up to 300 pM. The detection limit is 0.7 pM.

Keywords: Bilirubin, Enzyme electrode, Bilirubin oxidase

1. Introduction

Bilirubin (BR) is a tetrapyrrolic product of catabolism and the principal pigment of bile. The clinical importance of BR analysis is related to diagnosis and prediction of liver diseases as well as hemolytic disorders in adults and in newborn infants [ I , 21. In newly born infants in particular measurement of bilirubin is required for the prevention of neurologic abnor- mality and sensorineural hearing.

The determination of bilirubin is usually based on either diazo reaction or direct spectrophotometry [3-5]. Some electroana- lytical approaches have been reported for the determination of bilirubin. A polarographic method has been described by Koch and Akingbe [6]. Saar and Yarnitzky [7] suggested the use of a fast scan square wave voltammetry with a mercury working electrode. Wang et al. [8] described another voltammetric technique based on adsorptive stripping.

Few enzymatic methods have been described since the isolation and purification of bilirubin oxidase (EC 1.3.3.5; BOX) from Myvothecium verrucaria MT-1 [9].

Doumas et a/ . [ 101 proposed a spectrophotometric-enzymatic method for measuring bilirubin and Wang and Ozsoz [Il l reported a graphite-epoxy electrode incorporating bilirubin oxidase and horseradish peroxidase. The development of a self- assembled monolayer of cystamine as base layer for immobil- ization of BOX was presented by Riklin et al. [12].

This article provides a new contribution to the relatively few publications on the enzymatic-amperometric determination of BR. The construction of the modified hydrogen peroxide electrode with BOX is simpler and faster than the above mentioned methods [ 1 I , 121. The measurements are sensitive and the response time is fast.

2. Experimental

2.1. Materials and Apparatus

The hydrogen-peroxide-based platinum electrode (0.3 mm2 nominal surface area), with a built-in silver/silver chloride reference electrode, and the Amperometric Biosensor Detector, Model 3001, were from Universal Sensors Inc. (Metairie, LA). The current output was recorded with a REC 80 Servograph, Radiometer Copenhagen (Copenhagen, Denmark) X - t recorder.

The membranes used were: Immobilon A G affinity mem- brane, 0.65 pm pore size, 125 Irm thick (Millipore) and cellulose acetate, approximately MW cut-off = 100, prepared according to Mascini's et a/. method [13].

Bilirubin oxidase from Myrothecium verrucaria (EC 1.3.3.9, bovine serum albumin (BSA), aqueous glutaraldehyde (Grade IJ), indoxyl-3-sulfate (Indican), human hemoglobin and bili- rubin from bovine gall stones were purchased by Sigma (St. Louis, MO). The lyophilized BOX, containing ca. 25U of enzyme activity, was reconstituted with 0.5 mL of water. Each day, 1.0 x 1Op3M of bilirubin solution was prepared by dissolving 5.8 mg of substrate in 0.3 mL 0. I M sodium hydrox- ide, and diluting to lOmL with buffer solution. Dulbecco's physiological buffer (DPBS), pH 7.4, used in the experiments contains the following salt concentration: 137 mM NaCI, 2.7 mM KCl, 8.0 mM Na2HP04 and 1.5 mM KH2P04.

All reagents used were of analytical grade and all measure- ments were carried out at room temperature (23°C).

2.2. Electrode Assembling

The BOX membrane was prepared by crosslinking the enzyme with BSA and glutaraldehyde and then covalently bonding on preactivated membrane.

Approximately 1 mg of BSA was dissolved with 25pL of reconstituted bilirubin oxidase and 5 pL of glutaraldehyde (0.25% aqueous solution). The solution, gently mixed for a few seconds, was placed in the center of lcm2 Immobilon AV membrane. This was air-dried for I .5 h then washed with 0.1 M glycine solution, to eliminate the excess of glutaraldehyde, and DPBS.

The BOX membrane was placed over the cellulose acetate membrane previously fixed over the electrode jacket. The two membranes were then secured with an O-ring and a parafilm.

The jacket was filled with DPBS as internal solution and the platinum electrode was inserted and screwed down until the tip of the platinum electrode was firmly in contact with the cellulose acetate membrane. The platinurn working electrode was then poised at a constant potential of + 650 mV (versus the silver/ silver chloride internal reference).

2.3. Procedure

The bilirubin electrode was immersed in a l0mL beaker containing 5mL of DPBS stirred at a moderate speed with a

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230 A . Fovtunev, G. G. Guilbault

390

360

330

4 a ti-

9 300

B 0

270

240

210

-

-

-

-

-

I I i I I I I I 1.5 8 8.5 9 9.5 10

BOX (i.e., bacterial attack). This three membrane configuration showed a loss in sensitivity: bilirubin (M.W. 584.7) is probably Bilirubin + O2 --f Biliverdin + H202

The hydrogen peroxide produced is detected by the platinum electrode held at + 650 mV applied potential (vs. Ag/AgCl). The output current is therefore proportional to the bilirubin concentration.

The cellulose acetate membrane filters out most compounds

unable to diffuse through the polycarbonate membrane.

3.1. p~ Studies

The profile of bilirubin electrode attained at a concentration

1600

bilirubin concenlralion, yM Fig. 2. Calibration plot for the bilirubin eiectrode in DPBS.

Eli~clrounulwir 1996, 8, No. 3

Enzyme Electrode for the Determination of Bilirubin 23 1

I 1

c

a, 31 c

a, 31

Time

Fig. 3. Current-time recording upon increasing the bilirubin concentra- tion in I x lo-' M steps. The average value of 10 injections is 38 pA (RSD = 3.8%).

of bilirubin 1 x lop4 M is shown in Figure 1. The high response between pH 7.4 to 8.0 was obtained using phosphate buffer. For higher p H values we used a glycine buffer 0.02 M.

The assay performed at p H 7.0 shows a current output 37% lower of that obtained a t pH 7.4. This is due to partial aggregation into micelles of BR molecules not available for enzymatic oxidation [14]. Since it is reported that aqueous solutions of BR are less stable above pH 9.0 [15] we selected a DPBS buffer, p H 7.4, for all further experiments as a compromise between analytical sensitivity and stability.

3.2. Analytical Characteristics of the Electrode

A typical calibration plot for the bilirubin enzyme electrode is presented in Figure 2. The electrode response is linear within the concentration range 1-300 p M and a correlation coefficient r = 0.9997. The detection limit is 0.7pM. Figure 3 shows the reproducibility of the bilirubin probe for ten successive injections of BR 10pM (1 x IOp5M). The average value obtained is 38pA and the accuracy in terms of the relative standard deviation is 3.8%. The response time was approximately 2-3 min. The enzyme electrode was stored when not in use in

T'iblc 1 Response to BR and indic'in carried out using 0 5 mM of each compound Hcmoglobin d d e d 400 mg/L

Suhrtantr Keluri~e iut i~i ty 1x1

Bilirubin 100 Indican 584 Hemoglobin -

-~ ~~ _ _ ~~ ~

~~ ~~ - - -~

DPBS at 4°C. After 30 days we observed a 25% loss in sensitivity (Fig. 4).

3.3. Interferences Studies

Spectrophotometric determination of BR is affected by the presence of indican and hemoglobin [6, 161. Interference studies were performed by testing the response of the enzyme electrode to those substances.

As shown in Table 1 the presence of indican greatly affects the response of the enzyme sensor.

Concentrations of indican much higher than the amount used for this test (5 x lop5 M) are found in patients with renal fdilure, so as for spectrophotometric tests, a high value of BR must be suspected by the users of these procedures.

Interference from hemoglobin has been tested for recovery studies (Table 2).

As reported in selected literature, hemoglobin causes a decrease in BR values.

Table 2 Interference from hemoglobin in the en7ymatic-amperometric method

Bilirubrn udded Hemoglobin Biliruhtn found Rec o ve r j

~~ ~ ~.

[PMI [mglLl [ M I [ % I

5 0 50 5 0 100 10 0 9 9 99 5 0 100 4 6 92

10 0 9 4 94 5 0 300 3 5 70

10 0 7 1 71 -~ ~ ~~

Fig.

time, days 4. Long-term Stability of the bilirubin electrode when storcd in DPBS at 4 C.

232 A . Fovtunev. G. G. Guilbault

4. Conclusions

Even though our experiments were investigative in purpose and not optimized for analytical measurements we found the enzymatic-amperometric approach quite sensitive. Addi- tional studies have to be done to apply this method to reliable clinical measurements on physiological fluids. In particular, it is necessary to study the effect of other potential interferences, the correct preparation and storage of the BR standard (quite photosensitive) and comparison with standard methods.

5. Acknowledgement

A. Fortuney is grateful for the research-fellowship received from the University of Venice “Ca’ Foscari”, Italy.

6. References

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[S] B.W. Perry, B.T. Doumas, Clin. Cheni. 1983, 2Y, 297. [h] T.R. Koch, 0.0. Akingbe, Clin. Chem. 1981, 27, 1295. [7] J . Saar, C. Yarnitzky, Isr. J . Chem. 1983, 23, 249. [S] J . Wang, D. B. Luo, P.A.M. Farias, J . Electroanal. Chem. 1985, 185, 61. 191 S. Mu]-ao, M. Tanaka, Aguic. Bid . Chem. 1982, 46, 2031.

[lo] B.T. Doumas, B.W. Pcrry. B. Jendrzcjczak, L. Davis. C h . C h m . 1987,

[ I I ] J. Wang, M. Ozsoz, E/ectroanul.vJis 1990, 2, 647. [I21 A. Riklin, B. Shoham, Y. Migron, I . Willncr, Proc. Third World Congr.

Biosms., 1-3 June 1994, New Orleans. USA. Elsevier, Oxford 1995. [I31 M. Mascini, F. Mazzei, D. Moscone, G . Calabrese, M. Massi Benedetti,

Clin. Chew?. 1987, 33, 591. [14] R. Brodersen, J . Thielgaard, Scan. J . Clin. Lab. Invest. 1969, 24, 395. [ lS] J.D. Ostrow, L. Hammaker, R . Schmid, J . Clin. Invest. 1961, 40, 1442. [I61 R. Poon, H.I. Hinberg, Clin. Chem. 1985, 31. 92.

1973, IY. 984.

33, 1949.