A Miniature Palladium- Palladium-Oxide Enzyme Electrode for Urea Determination

Neil J. Szuminsky* and Alice K. Chent Program in Medical Technology, School of Health Related Professions, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

C. C. Liu Chemical Engineering Department, Case Western Reserve University, Cleveland, Ohio 44106

Accepted for Publication October 22, 1983

Enzyme electrodes have provided simple, essentially reagentless, substrate analyses which are both specific and sensitive. The development and usage of urea electrodes for analytical and clinical applications have been reviewed in several Enzyme electrodes for urea deter- mination are usually prepared by immobilizing urease with a potentiometric gas sensor, such as the NH3 or C02 gas electrode, or an ion-selective electrode, namely the pH or ammonium ion-selective electrode.

In addition to conventional glass pH electrodes, many metal-metal-oxide pH sensors have been developed. Most of these have limited applications due to unstable re- sponses or relatively short lifetimes. However, an anti- mony metal electrode and an iridium dioxide electrode were used successfully for urea electrode^.^^^

Recently, a wire-form palladium-palladium-oxide (Pd-PdO) pH electrode was d e ~ e l o p e d . ~ , ~ The Pd-PdO electrode reacts to H+ ion according to the reaction:

PdO + 2H+ + 22- =+ Pd + H 2 0

According to the Nernst equation, the zero-current elec- trochemical potential of this reaction can be used for pH measurement. The oxide layer on the palladium wire could be produced electrochemically in a molten salt6 or by chemical oxidation at high temperature^.^ These wire- form pH electrodes are miniature in size, have excellent pH response, and a relatively very long lifetime. They are most useful for aqueous solutions in the pH range 3-11 and provide a promising alternative to the glass pH elec- trode as the sensor for enzyme electrodes. Applications of the Pd-PdO electrode in sensors for C02 and pH measure- ment has been r e p ~ r t e d . ~ . ~ This communication describes the preparation and evaluation of urea electrodes based on the combination of immobilizing urease and the wire-form miniature Pd-PdO pH electrode.

*Present address: Med-Chek Laboratories Inc., 1386 Old Freeport

tTo whom all correspondence should be addressed. Road, Pittsburgh, Pennsylvania 15238.

Biotechnology and Bioengineering, Vol. XXVI, Pp. 642-645 (1984)

EXPERIMENTAL

Apparatus and Reagents

The zero-current potentials of the Pd-PdO electrode and the enzyme electrode were measured against a stan- dard saturated calomel electrode (SCE) using an Orion 701A digital pH meter which was interfaced with a Radio Shack TRS-80 microcomputer. In addition to data collec- tion, the computer was programmed to plot the electrode response on a strip-chart recorder.

Buffers, urea standards, and other solutions were made in distilled water with analytical grade reagents from Fisher Scientific Company. A combination glass pH elec- trode (Fisher Scientific Company, No. 13-639-104) was used to determine the pH of the buffers. The enzyme urease (E.C.3.5.1.5, type VII, powder from Jack Beans, 53 IU/mg), bovine serum albumin (BSA), and glutaralde- hyde (25% in water) were obtained from Sigma Chemical Company (St. Louis, MO).

Electrode Preparation and Testing

The fabrication of the Pd-PdO pH electrode can be de- scribed as follows. A palladium wire (95% pure, Mathey Bishop Inc.), 0.75 mm in diameter and 5 cm in length, was pushed through a glass tubing ( 1 .O mm i.d.) with 1.5 cm of the wire extended at one end of the glass tubing. The glass tubing was then sealed around the wire by heating in a flame. The exposed wire was then electrochemically ox- idized in molten salt according to the procedure of Liu et a1.6 All Pd-PdO electrodes were conditioned by soaking in phosphate buffer, O.lM, pH 7.0, for a minimum of 3 h prior to pH tests. Between tests, the Pd-PdO electrodes were stored in the same buffer solutions. These electrodes were tested for their pH response in phosphate buffer over the pH range 5.4-9.5. With careful control of the oxida- tion conditions, electrodes with reproducible responses were produced. Only electrodes with slope of 53 to 65 mV

0 1984 John Wiley &Sons, Inc. CCC 0006-35921841060642-03$04.00

were used in the making of the urea electrode. The en- zyme, urease, was immobilized to the Pd-PdO electrode by crosslinking the enzyme with BSA using glutaralde- hyde with modification of the procedure of Mascini and Guilbault.lo A urease-BSA solution was prepared by dissolving 10 mg urease and 15 mg BSA in 100 pL 0.1M phosphate buffer, pH 7.0. Five microliters of this urease-BSA solution was mixed with 3 pL of 25% (w/v) glutaraldehyde. One microliter of this mixture containing 3.2 units of urease was then quickly coated evenly over the surface of the Pd-PdO electrode using a Drummond Wire-trol pipette. After 1 min, a second coating of the urease mixture was applied to the electrode; this coating was allowed to polymerize at room temperature for 10 min. The electrodes were then washed first for 15 min in distilled water to remove free unreacted glutaraldehyde and then 15 min in 0.1M glycine to inactivate any remain- ing glutaraldehyde. The enzyme electrodes were stored at 4°C in 0.1M phosphate buffer, pH 7.0, containing 1.0 X 10W3M ethylenediaminete-tetraacetate (EDTA).

All potentiometric measurements were conducted at 25°C in a 50-mL beaker with a magnet stirred at 200 rpm. The urea electrodes were tested in phosphate buffers con- taining 0.1M NaCl and 1.0 X 10W3M EDTA. The urea electrodes were equilibrated first in a 0.1M phosphate wash buffer until a stable potential was obtained and then transferred to 50 mL of the test buffer containing urea. The pH difference between the wash buffer and the test buffer was maintained at less than 0.05 pH unit. The po- tential difference between the wash buffer and the test buffer was then used to plot the calibration curve.

RESULTS AND DISCUSSION

The pH responses of three Pd-PdO electrodes in 0.1M phosphate buffer over the pH range 5.6-9.5 are shown in Figure 1. With some modification of the oxidation condi- tions, we were able to reduce the super-Nernstian slope of 71 mV/pH unit reported earlier6 to a range of 53-65 mV/pH unit. The variations in slope could have been caused by composition variation of the molten salt, such as the nitrate content, and the variation in the oxidation conditions used. The time response of a typical Pd-PdO electrode is shown in Figure 2. Within two minutes of im- mersing the electrode into the test buffer, 98% of its steady-state value was usually obtained. The response times of the electrodes were found to be sensitive to air dry- ing and the response time increased up to five minutes with reproducible voltage.

As shown in Figure 1, the enzyme-BSA coating did not change the overall pH response of the electrode as deter- mined by the slope, steady-state potential or response time. Calibration curves of the urea electrodes are shown in Figure 3. In general, the response of the urea electrode exhibits characteristics similar to those reported ear- lier.1,4*10,11 Using a 1.0 X 10W2M phosphate buffer, at pH 7.0, a linear response was obtained over the urea concen- tration range from 2.0 X 10-4M to 5.0 X 10V3M with a

2%

200 > E

100

6.0 7.0 8.0 9.0 PH

Figure 1. pH response of the ( A , u, v ) Pd-PdO pH electrodes and ( 0 )

urea electrode (made from the Pd-PdO pH electrode as shown in [ I ) in 1.0 X 10-'M phosphate buffer.

> E -100 1 x -40

-20

oq -5 -4 -3 -2

10 10 10 10 LO UREA CONCENTRATION, M

Figure 2. phosphate buffer.

Response times of Pd-PdO pH electrodes in 1.0 X 10K'M

slope of 52.6 mV. Since the sensitivity of most enzyme elec- trodes is determined by enzyme activity, comparison of the slope to the pH sensitivity of the Pd-PdO electrode and the theoretical Nernstian prediction indicates that the urease activity on the electrode was quite sufficient.

Full response of the urea electrode was obtained within five minutes after the electrodes were transferred from the wash buffer to the test buffer. The response characteristics of the urea electrodes were found to be very sensitive to de- tails involved in urease-BSA coating of the Pd-PdO pH electrode. As shown in Table I, the urea electrodes gave re- producible responses with coefficients of variance ranged from 3.3 to 7.9%.

In addition to the urease activity, the detection limits and sensitivity of urea electrodes based on pH sensors also

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300 I I I '

100

-5 -4 -3 -2 -1 10 10 10 10 10

UREA CONCENTRATION. n

Figure 3. Calibration curves of the urea electrodes in 1.0 X lO-'M phosphate buffer with 1.0 X 10W'M NaCl and 1.0 X 10-3M EDTA at ( A ) pH 6.5, (0) pH 7.0, and ( 0 ) pH 7.4.

DH 4.0 - I I I

Table I. phosphate buffer, 1 X 10W'M NaCI, and 1 X 10-3M EDTA).

Reproducibility of urea electrode response (in 1 X lOP2M

Urea concentration

~ o - ~ M 1 0 - 3 ~ lo-2M 3 x lo-2M Electrode (mV) (mV) (mV) (mV)

218 -21 - 51 - 108 - 1 1 1 225 - 19 -64 -99 - 103 226 - 18 - 66 - 107 - 112 228 - 22 -6s - 104 - 1 1 1 229 - 20 - 69 - 105 -110

Mean k SDa -2O.Ok1.6 - 6 4 . 2 k 4 . 4 -1Mf3.5 -109 .4k3.7 C.V. 7.9% 6.9% 3.4% 3.3%

aSD refers to standard deviation.

depend on the pH and buffer capacity of the test media." The effect of buffer pH on the electrode response was studied by measuring the electrode response to urea in phosphate buffer at constant buffering strength. As shown in Figure 3, at lower pH, the linear portion of the calibration curve at higher urea concentrations was ex- tended. This occurs because the enzyme reaction involves the formation of self-buffering products, one bicarbonate and two ammonium ions, which form a buffer with a pKa of 8.83. As the urease reaction proceeds, the internal pH of the enzyme electrode begins to approach the pKa of the products. With a more acidic buffer, a larger pH change occurs before the pKa can be reached. This results in super-Nernstian slope of 81 mV at pH 6.5 and extension of the calibration curve to higher urea concentrations. At urea concentrations less than 1.0 X 10-3M, the self- buffering become less effective and the response of the en- zyme electrode was essentially pH independent.

The effects of buffer strength on the calibration curve at constant pH are shown in Figure 4. As expected, the elec- trode response for a given urea concentration was very sen- sitive to the buffering capacity of the test solutions. The rate of H+ ion consumption at any given urea concentra-

OH 5 . 6

250 t I DH 6 . 4 >

200 2

Q r L Y r 0

a 150

tion is determined by the diffusion of urea into the enzyme layer, the enzyme activity and the buffer strength and pH at the surface of the Pd-PdO pH electrode. Therefore, a greater pH change was detected as buffering strength de- creased and lower buffer capacity increased the response as well as the linearity of the urea electrode. At pH 7.4, the calibration curves for 1.0 X 10W3M and 1.0 X 10W2M phosphate test buffers gave reproducible slopes. Higher buffer capacity shortened the linear range at the lower urea concentration range. These results agree well with other pH-based urea electrodes reported in the litera- ture. 1 t 4 7 1 1

The urea electrodes were found to be stable for more than three weeks when 1 .O X 10W3M EDTA was added to the test and storage buffers. The pH response of the Pd-PdO electrode was not altered by storage in EDTA so- lutions for periods of up to six weeks. By chelating heavy metal ions which are inhibitors of urease, EDTA stabilized the enzyme activities of the immobilized urease.

The pH response of the Pd-PdO electrode was found to be very sensitive to reducing substances: ascorbic acid, NADH, hydrazine, and strong redox complex such as ferro and ferric cyanide. The reducing substances caused the potential of the Pd-PdO electrode to shift hundreds of mV with loss of the typical pH response. Similar obser- vations were reported by Grubb and King.7 For routine applications, reducing and other interfering substances could be excluded by coating the Pd-PdO electrode with membranes permeable to hydrogen ions only. The effect of specimen on the test buffer pH can be reduced to a negligi- ble amount by high dilutions of the specimen and applica- tion of a dual-sensor device using an auxiliary Pd-PdO pH electrode as reference.

In conclusion, the feasibility of using the wire-form Pd-PdO pH electrode for enzyme electrode application was demonstrated by the successful development of an urea electrode. This urea electrode produced a typical sig-

644 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 26, JUNE 1984

moidal response curve to urea in aqueous solutions. The linear range could be varied by adjusting the pH and the buffer capacity of the testing buffer. The advantages of this wire-form, pH-based urea electrode include minia- ture size, simple fabrication, and the possibility of opti- mizing test conditions for specific applications.

The financial support of this work by the Health Research and Service Foundation, under Grant No. U-52, is gratefully acknowledged.

References

1. R. K. Kobos, in Ion-Selective Electrodes in Analytical C h e m i t y , H. Freiser, Ed. (Plenum, New York, 1980), Vol. 2. p. 31.

2. R. L. Solsky, CRC Crit. Rev. Anal. Chem., 14, 1 (1982). 3. M. E. Meyerhoff and Y. M. Fraticelli,Anal. Chem., 54,27R(1982). 4. P. W. Alexander and J. P. Joseph, Anal. Chim. Acta, 131, 103

5. R. M. lanniello and A. M. Yacynych, Anal. Chim. Acta, 146, 249

6. C. C. Liu, B. C. Bocchiocchio, P. A. Overmyer, and M. R. Neuman,

7. W. T. Grubb and L. H. King, Anal. Chem., 52, 273 (1980). 8. R. L. Coon, N. C. Lai, and J. P. Kampine,J. Appl. Physiol., 40,625

9. C. C. Liu and M. R. Neuman, Diabetes Care, 5, 275 (1982). 10. M. Mascini and G. Guilbault, Anal. Chem., 49, 795 (1977). 11. N. Nilsson, A. Akerlund, and K. Mosback, Biochim. Biophys.

( 1981 ).

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Acta, 320,529 (1973).

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