thin-film antimony–antimony-oxide enzyme electrode for penicillin determination

7
Thin-Film Antimony-Antimony-Oxide Enzyme Electrode for Penicillin Determination M. T. Flanagan and N. J. Carroll Department of Electronic and Electrical Engineering, University College London, Torrington Place, London, WC1E 7JE, England Accepted for publication August IS, 7985 A potentiometric penicillinase electrode is reported in which the base pH transducer is a thin-film anti- mony-antimony-oxide electrode deposited by vacuum evaporation. Several enzyme immobilization procedures have been examined and a crosslinked protein film found to be the most appropriate to this type of sensor. The use of an adjacent antimony-antimony-oxide track as a pseudoreference electrode was successfully demon- strated. The overall response was shown to be indepen- dent of the stirring rate above 100 rpm, but the kinetics of the response were found to depend markedly on the stirring rate. The intrinsic linear response range was 3 x W4M to 7 x '10-3M penicillin G. Linearizing transforms that extend the useful range were examined. INTRODUCTION The immobilization of enzymes onto base trans- ducers that monitor one of the components of the cat- alyzed reaction has proved a useful way of fabricating reagentless sensors in the laboratory.' However, few such enzyme electrodes have successfully reached the wider arena of process control and commercial medical diagnostics. The development of sensors for these ap- plications will be facilitated by designs which will allow the simultaneous monitoring of primary analyte and interferences enabling correction of the primary signal whilst retaining inexpensive manufacturing techniques and easy replacement of the sensor head. Miniaturi- zation is also desirable in order that sample volumes may be reduced. The limited lifetime of the immobilized enzyme has led to several workers seeking inexpensive alternatives to the commercial electrodes, e.g. pH and p02 elec- trodes, used as the base transducers. Several metal-metal oxide pH electrodes have been developed and some have proved acceptable as base transducers. Urease has been immobilized on antimony,2 i r i d i ~ m , ~ and palladi~m.~ Liu and Newmans have also fabricated thin-film palladium-palladium-oxide pH electrodes and have suggested their use as more convenient base transducers. Ion-sensitive field-effect transistors have been used as miniature base transducers in potentio- metric enzyme electrodes (chemFET~).~.' The poten- tial that ChemFETs offer for the fabrication of multi- gate sensors for complex solutions, e.g. fermentation broths, in which both multiple analytes and interfer- ences may be simultaneously monitored is equally at- tractive.8 An analogous multigate sensor in which the problems of integrating immobilization techniques with semiconductor device fabrication are avoided would be even more attractive. The successful use of cast antimony rod as a base transducer suggests a way in which this might be achieved. Antimony may be deposited by vacuum evaporation as thin films,9 though such films have never been ex- amined as pH electrodes. The combination of vacuum evaporation with the use of masks will enable the pro- duction of small inexpensive multitrack anti- mony-antimony-oxide electrodes. In this article, we describe the preparation of thin-film anti- mony-antimony-oxide electrodes, their pH sensitivity, their stability in electrolyte solutions, and their use as the base transducers in penicillin-sensitive enzyme electrodes. EXPERIMENTAL Reagents and Apparatus PeniciUinase (penicillin amido-/34actamhydrolase, EC 3.5.2.6, Sigma type 1 from Bacillus cereus, 320 unitshg solid), bovine serum albumin, penicillin G (benzylpen- icillin, potassium salt), glutaraldehyde (25% aqueous solution), l-cyclohexyl-3-(2-morpholinoethyl)-carbo- diimide metho-p-toluenesulphonate, and 3-aminopro- pyltriethoxysilane were obtained from the Sigma Chemical Company (Poole). Vinyltrichlorosilane and triphenylsilylchloride were obtained from Fluka AG (Buchs) and Aldrich (Gillingham), respectively. Anti- mony powder (not less than 98.5%) was obtained from BDH (Poole). Potentiometric measurements were made with a Corning pH meter 130 used in its millivoltmeter mode Biotechnology and Bioengineering, Vol. XXVIII, Pp. 1093-1099 (1986) Q 1986 John Wiley & Sons, Inc. CCC 0006-3592/86/071093-07$04.00

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Thin-Film Antimony-Antimony-Oxide Enzyme Electrode for Penicillin Determination

M. T. Flanagan and N. J. Carroll Department of Electronic and Electrical Engineering, University College London, Torrington Place, London, WC1E 7JE, England

Accepted for publication August IS, 7985

A potentiometric penicillinase electrode is reported in which the base pH transducer is a thin-film anti- mony-antimony-oxide electrode deposited by vacuum evaporation. Several enzyme immobilization procedures have been examined and a crosslinked protein film found to be the most appropriate to this type of sensor. The use of an adjacent antimony-antimony-oxide track as a pseudoreference electrode was successfully demon- strated. The overall response was shown to be indepen- dent of the stirring rate above 100 rpm, but the kinetics of the response were found to depend markedly on the stirring rate. The intrinsic linear response range was 3 x W 4 M to 7 x '10-3M penicillin G. Linearizing transforms that extend the useful range were examined.

INTRODUCTION

The immobilization of enzymes onto base trans- ducers that monitor one of the components of the cat- alyzed reaction has proved a useful way of fabricating reagentless sensors in the laboratory.' However, few such enzyme electrodes have successfully reached the wider arena of process control and commercial medical diagnostics. The development of sensors for these ap- plications will be facilitated by designs which will allow the simultaneous monitoring of primary analyte and interferences enabling correction of the primary signal whilst retaining inexpensive manufacturing techniques and easy replacement of the sensor head. Miniaturi- zation is also desirable in order that sample volumes may be reduced.

The limited lifetime of the immobilized enzyme has led to several workers seeking inexpensive alternatives to the commercial electrodes, e.g. pH and p 0 2 elec- trodes, used as the base transducers. Several metal-metal oxide pH electrodes have been developed and some have proved acceptable as base transducers. Urease has been immobilized on antimony,2 i r i d i ~ m , ~ and pa l l ad i~m.~ Liu and Newmans have also fabricated thin-film palladium-palladium-oxide pH electrodes and have suggested their use as more convenient base transducers. Ion-sensitive field-effect transistors have been used as miniature base transducers in potentio-

metric enzyme electrodes (chemFET~) .~ . ' The poten- tial that ChemFETs offer for the fabrication of multi- gate sensors for complex solutions, e.g. fermentation broths, in which both multiple analytes and interfer- ences may be simultaneously monitored is equally at- tractive.8 An analogous multigate sensor in which the problems of integrating immobilization techniques with semiconductor device fabrication are avoided would be even more attractive. The successful use of cast antimony rod as a base transducer suggests a way in which this might be achieved.

Antimony may be deposited by vacuum evaporation as thin films,9 though such films have never been ex- amined as pH electrodes. The combination of vacuum evaporation with the use of masks will enable the pro- duction of small inexpensive multitrack anti- mony-antimony-oxide electrodes. In this article, we describe the preparation of thin-film anti- mony-antimony-oxide electrodes, their pH sensitivity, their stability in electrolyte solutions, and their use as the base transducers in penicillin-sensitive enzyme electrodes.

EXPERIMENTAL

Reagents and Apparatus

PeniciUinase (penicillin amido-/34actamhydrolase, EC 3.5.2.6, Sigma type 1 from Bacillus cereus, 320 unitshg solid), bovine serum albumin, penicillin G (benzylpen- icillin, potassium salt), glutaraldehyde (25% aqueous solution), l-cyclohexyl-3-(2-morpholinoethyl)-carbo- diimide metho-p-toluenesulphonate, and 3-aminopro- pyltriethoxysilane were obtained from the Sigma Chemical Company (Poole). Vinyltrichlorosilane and triphenylsilylchloride were obtained from Fluka AG (Buchs) and Aldrich (Gillingham), respectively. Anti- mony powder (not less than 98.5%) was obtained from BDH (Poole).

Potentiometric measurements were made with a Corning pH meter 130 used in its millivoltmeter mode

Biotechnology and Bioengineering, Vol. XXVIII, Pp. 1093-1099 (1986) Q 1986 John Wiley & Sons, Inc. CCC 0006-3592/86/071093-07$04.00

(relative mV setting) and connected to a chart recorder. A Pye-Unicam-Ingold calomel electrode (No. 303) was used in those experiments which required a commer- cial reference electrode. The pH meter was calibrated in its pH mode using a Pye-Unicam combination glass electrode (No. 401E7) and standard buffers. The glass electrode was then replaced by the anti- mony-antimony-oxide thin-film electrode and the me- ter switched to the millivoltmeter mode. No further adjustment was made to the meter when pH response curves were recorded. Consequently, the pH response curves presented all have the same instrumental offset.

Twenty-milliliter volume samples were used in the electrode testing. The penicillin was added to the test solutions as solution aliquots of 200 pL or smaller to give final concentrations of l O m M or less and as a 400- pL aliquot to give a final penicillin concentration of 201x184. The buffer concentrations are listed in the ap- propriate figure legends. The solutions were stirred with a 10 x 5 mm magnetic flea. The stirring rates quoted are the revolutions per minute of the magnetic flea and were measured stroboscopically.

Computation

Nonlinear regressions were performed using the sim- plex minimization procedure of Nelder and Mead. lo

The minimizations were constrained, where necessary, by the use of simplified penalty functions described elsewhere. I Polynomial fittings were performed using Gaussian elimination to solve the linear equations aris- ing from a least-squares procedure.I2 All computations were carried out on a Hewlett-Packard model 9825A microcomputer.

Electrode Preparation

Glass and ceramic substrates were cleaned by wash- ing with aqueous detergent, rinsing exhaustively with water, and sonicating twice in water for 20 min (Kerry Pulsatron 125 sonication bath). They were then im- mersed in 1M HCl for 10 min, rinsed with water, im- mersed in 1M NaOH for 10 min, rinsed exhaustively with water and finally rinsed with propan-2-01 and air- dried. The water was deionized and distilled.

Glass surfaces were derivatized with 3-aminopro- pyltriethoxysilane by the aqueous method of Weetall13 and with vinyltrichlorosilane or triphenylsilylchloride by immersion in 10% (w/v) solutions of these reagents in chloroform or toluene, respectively. After 5 h the latter substrates were rinsed with the above solvent and with acetone, air-dried, and placed in an oven at 115°C for 14 h.

Antimony films were deposited on alumina ceramic sheets, derivatized glass slides, and nonderivatized glass slides (Chance-Popper soda-glass microslides). These substrates were placed 8 cm above the source (anti-

mony powder in a molybdenum boat) in an Edwards E306 vacuum coater. In order to give the appropriate electrode pattern, aluminum masks were placed on the surface of the substrate. The antimony films were de- posited by upward evaporation at pressures below lop3 Pa at a deposition rate of 300 &s and with a heating current of 0.5 A. Film thicknesses were measured using a Talystep and typically were 3 pm.

Three procedures were examined in the immobili- zation of the penicillinase on the antimony thin-film surfaces. Entrapment in polyacrylamide, following the method of Papariello, Mukherji, and Shearer,14 was reinforced by coverage with Visking dialysis film. Crosslinking with bovine serum albumin via glutaral- dehyde (20 unitdmg protein) was achieved by the method of Mascini and Guilbault.I5 The crosslinked film formed a stable bond with the antimony film surface. A water- soluble carbodiimide coupling procedure l6 was used to obtain a covalent linkage of the penicillinase to the antimony film surface. In all cases an enzyme electrode response was only obtained if the antimony film was immersed in distilled water for at least 2 h prior to the enzyme immobilization. The color of the electrodes changed from silver-grey to matt black. It is probable that the essential hydrated oxide surface layer was formed at this point. The prepared electrodes were stood in O.01Mphosphate buffer, pH 7.0, for 5 h before use.

RESULTS AND DISCUSSION

pH Response

The pH-dependent electrical potential generated by the Sb-Sb203-electrolyte-reference-electrode cell is, to a first approximation, governed by the overall reactions .

Sb S Sb3' + 3e- (1)

Sbz03 + 3Hz0 2Sb3+ + 6 0 H - (2)

and

and is given by

E = E ~ o - k(pH) = Eo - klog(K,)

+ (k/6)10g(&) + Eref - k(pH) (3)

where K , is the autoprotolysis constant for wa- ter: ( a H + ) ( a o H - ) ; Kd is the equilibrium constant, (aSb3+)2(aOH-)6; ai is the activity of species i; Eo is the oxidation-reduction potential of antimony; and Eref is the half-cell potential of the reference electrode. l7 Pa- rameter k is the Nernstian gradient, RTlog(lO)/F, which equals 58.76 mV/pH unit at 23"C, the temperature a t which the measurements reported in this article were made.

Figure 1 shows the pH response of antimony thin

1094 BIOTECHNOLOGY A N D BIOENGINEERING, VOL. 28, JULY 1986

films, deposited on alumina substrates, in different buffer solutions over the relevant pH range for the subsequent enzyme electrode work. The response is linear over this range with an average gradient of -52.5 k 1.5 mV/pH unit. This is less than the Nernstian value re- quired by eq. (3) but is comparable with the value obtained for a commercial cast-rod antimony electrode (- 49.8 ? I .7 mV/pH unit) which was included for comparison. Antimony electrodes are rarely well be- haved,” e.g., the equilibrium represented by eq. (2) may be disturbed by the presence of metal-ion com- plexing agents thus shifting the value of EGO. Figure 1 shows that though ELo is, for practical purposes, independent of the phosphate buffer concentration, it is shifted by ca. 10 mV on replacing phosphate buffer by Tris buffer. The antimony films formed strong bonds with the alumina substrates and were not removed by continuous immersion in aqueous solutions for at least 20 days. The pH response curve did not vary through- out this period.

Immobilization of the penicillinase by the carbodi- imide-catalyzed covalent coupling of the protein to the hydrated antimony-oxide surface layer might be ex- pected to shift EGO by perturbing the equilibrium, eq. (2) and, possibly, by altering Eo. The value of ELo was shifted, after the carbodiimide immobilization proce- dure, by ca. -27 mV. The gradient was essentially unaltered, -54.0 2 4.6 mV/pH unit (Fig. 1) . Immo-

-300

> E -350- .- c

C 0) c

2 al B c 4 -400- Lu

I \ I I i I i I I I

.

-450 c \-I 6.0 7.0 8.0 9.0

PH

Figure 1. The pH response of antimony-antimony oxide thin film electrodes in (+) 0.001M, (.) 0.01M, and (0)O.lMphosphate buffer and in 0.09M (0) Tris buffer and of a (A) “glutaraldehyde” and a (0) “carbodiimide” penicillinase thin-film antimony-antimony ox- ide electrode, both in 0.01M phosphate buffer. A commercial cast- rod antimony electrode (EIL) response in 0.1M phosphate buffer was included (V) for comparison. The reference electrode was the standard calomel electrode and the stimng rate was 390 rpm.

bilization of the penicillinase by glutaraldehyde cross- linking should not result in any major chemical mod- ification of the antimony oxide surface layer and hence might be expected to leave the pH response unaltered. This was found to be the case (Fig. 1). The value of ELO was not shifted and the gradient was - 51.3 (? 0.2) mV/pH unit.

Antimony films deposited onto nonderivatized glass substrates floated off on immersion in aqueous solu- tions. Those deposited onto derivatized glass sub- strates did not float off on immersion in aqueous so- lutions but did develop pinholes of ca. 0.4 mm diameter and of density ca. 5 cm-2. The pH responses of these films were nonlinear. The gradients vaned by up to 18 mV/pH unit across a pH unit. There was no obvious correlation between the nature of the derivatization and the stability or the pH response. The appearance of the pinholes may arise from an incomplete deriva- tization of the glass surface. The precise determinants of the efficiency of the derivatizations have yet to be fully elucidated. l 3 All the subsequent enzyme electrode work was carried out on antimony films deposited on alumina substrates.

Penicillin Response

Figure 2 shows the response to penicillin of the pen- icillinase antimony thin-film electrodes in which the three different immobilization procedures were ex-

120 , 1 1 I 1

I I I 1 10’~ 16 162

Penicillin G Concentration, M

Figure 2. Calibration curves for (a) “glutaraldehyde,” (b) “car- bodiimide,” and (c) “polyacrylamide” penicillinase antimony- antimony oxide thin-film electrodes in (.) 0.001M, (A) 0.01M, and (+) 0.1M phosphate buffers, at pH 7.0. The reference electrode was the standard calomel electrode and the stirring rate was 390 rpm.

FLANAGAN AND CARROLL: THIN-FILM ENZYME ELECTRODE 1095

amined. The reference electrode was the standard cal- omel electrode. The change in potential is the differ- ence between the value before the addition of the penicillin to a pH 7.0 phosphate buffer solution and the maximum potential reached after the addition. The “carbodiimide” and “glutaraldehyde” electrodes showed responses typical of more conventional peni- cillinase electrodes. 14~18-21 The “polyacrylamide” elec- trode showed a poor response which is consistent with the failure of the polyacrylamide to form a strong bond with the antimony oxide surface layer. A covering di- alysis film was used to keep the polyacrylamide film in place. Figure 2 also shows the strong dependence of the response of the electrodes on the concentration of buffer in the analyte solution. This is typical of pH- based potentiometric enzyme electrodes.18.20 The buffer competes with the base transducer for the protons re- leased by the catalyzed reaction and hence the re- sponse of the electrode falls with increasing buffer con- centration. The fall in response, at high penicillin concentrations, may be due to product inhibition. Pen- icilloic acid is an inhibitor of penicillinase with a K I of ca. 40mM for the free enzyme.22

Figure 3 shows the mean response and “between electrode errors” for “glutaraldehyde” electrodes of dimensions 15 x 25 mm. Also shown is the response of a 5 x 5 mm “glutaraldehyde” electrode in which the curve does not differ greatly from that of the larger electrodes. This should be the case for a steady-state measurement in a potentiometric sensor and indicates that there should be few problems on miniaturization. Antimony electrodes are not used routinely for pH measurements as their responses are dependent on too many parameters in addition to pH, e.g., the buffer ion present as reported in this article. Consequently, a

I I I I I

I 1c5 lo4 10

Penicillin G Concentration, M

Figure 3. Calibration curves for 25 x I5 mm “glutaraldehyde” electrodes (symbol 0 shows the mean value, the error bars represent the “between electrode errors”) and a 5 x 5 mm (m) “glutaralde- hyde” electrode referenced against the standard calomel electrode and for a 5 x 5 rnm “glutaraldehyde” electrode referenced against a 5 x 5 mm antimony-antimony oxide thin-film (A). The buffer was 0.001M phosphate, pH 7.0, and the stirring rate was 390 rpm.

thin-film antimony enzyme electrode will only make sense if the standard reference electrode is replaced by an antimony thin-film electrode onto which no en- zyme has been immobilized. The economic fabrication of miniature multigate devices also dictates such a change. The response of an antimony thin-film enzyme electrode/antimony thin-film pseudoreference elec- trode couple will only be independent of interfering potential changes, occumng at the base transducer level, if the response of the enzyme electrode to pH (and other interfering ions) is identical to that of the pseu- doreference electrode, i.e., if the immobilization pro- cedure does not change the response curve. It has been shown (Fig. 1) that the glutaraldehyde procedure sat- isfies this criterion. Figure 3 shows the response of a 5 x 5 mm “glutaraldehyde” electrode in which the reference electrode was also a 5 x 5 mm antimony film deposited on the same alumina substrate. The re- sponse curve did not differ significantly from that in which the reference electrode was the standard calo- mel electrode.

If the penicillinase electrode is to be used over a narrow range of penicillin concentrations in the range 3 x 10-4M to 7 x 10-3M, the response may be treated as being linear. If the sensor is to be used without further modification of its design over a more extended range or outside these limits, a more sophisticated anal- ysis of the data, to be performed by an associated microprocessor, will be required. In order to determine an appropriate analysis we first examined the possi- bility of fitting the data to the polynomials:

n

v = x UjS’ i = O

(4)

and n

v = 2 Ujlog’(S) ( 5 ) i = O

where V is the potential change, S is the concentration of penicillin and n is the degree of the polynomial. Table I shows the results of fitting a typical “glutar- aldehyde” electrode response to equations 4 and 5 . The usefulness of these equations as interpolating polynomials may be judged from the standard error of the fit,

C [V(experimental) i = 1 r - V(cal~ulated)]~/(m - n - 1)

i“ where m is the number of data points. Inspection of Table I shows that acceptable interpolating polyno- mials are only obtained for high-degree polynomials, e.g. n = 5. This may allow the use of this technique

1096 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 28, JULY 1986

Table I. The fitting of the response of a “glutaraldehyde” electrode to several polynomials (data as in Fig. 4).

~~~ ~ ~ ~

Standard error of the fit (as a percentage of the Degree of

Polynomial polynomial mean value of V ) ~~~~~~

Equation (4) 1 2 3 4 5

2 3 4 5

Equation ( 5 ) I

~

8.601 6.756 2.565 2.349 0.046

13.01 4.79s 4.137 1.323 0.002

(22.2) (17.4) (6.6) (6.1) (0.1)

(33.6) (12.4) (10.7) (3.4) (0.005)

in specialized applications where the required accuracy would warrant the trouble of taking five experimental calibration points. However, for more routine assay work, n = 5 is too large. Consequently, the following transforms of the response data have been examined: a) log[(V, - V)/v] vs. log(S); b) 1/V vs. U S ; and c) V/S vs. S , where V, is the potential change at a pen- icillin concentration of IOmM. As penicillinase, to a first approximation, obeys Michaelis-Menten kinetics, and if V and V , were equated to the rate and maximum rate of the catalyzed reaction, the above transforms would be linear if applied to the enzyme in free solu- tion. They will not necessarily be so in the case of the immobilized enzyme in which the kinetics are made complex by the introduction of diffusion terms and base transducer kinetic terms and in which V, has been redefined, as above, for operational simplicity (see Figs. 2 and 3). In practice transform a proved a useful li- nearizing transform for both “glutaraldehyde” and “carbodiimide” electrodes. Figure 4 shows a typical transform for which the correlation coefficient was - 0.989. The coefficients for transforms b and c applied to the same data were 0.973 and 0.876, respectively. The success of transform a is in keeping with its use- fulness in analyzing complex biological equilibria else- where, e.g., as a Sips plot transform in analyzing het- erogeneous antibody-antigen e q ~ i l i b r i a . ~ ~ In the present context, it represents a three-point calibration curve: V, plus two points to establish the gradient and intercept.

Figure 5 shows the lifetime obtained for “carbodi- imide,” “glutaraldehyde,” and “polyacrylamide” electrodes. A typical batch fermentation period is also shown to indicate the minimum lifetime that will ulti- mately be required of any penicillinase electrode used in penicillin fermentation process control. The “car- bodiimide” electrodes always decayed within two to three days. The functioning of an antimony-antimony- oxide pH electrode depends on the limited solubility of the antimony oxide layer [eq. (3)l. Consequently, the surface layer is continually, albeit slowly, eroded.

Penicillin G Concentration, M

Figure 4. A linear transform of the 5 x 5 mm “glutaraldehyde” electrode versus pseudoreference electrode data presented in Fig- ure 3.

As the “carbodiimide” coupling involves a covalent attachment to the hydrated surface oxide layer the im- mobilized enzyme layer will also be eroded giving rise to the observed short lifetime of the sensor. The “glu- taraldehyde” immobilization does not involve any such linkage and a stable electrode is observed when this procedure is used. We are still examining the origins of the decay after ten days. A thin film antimony pH sensor still functions after this time. The low response of the “polyacrylamide” electrode eliminates this as an electrode of interest.

h

I I I 1 1 A

0 0 100 200 300

Time. hours

Figure 5. The lifetimes of (A) “glutaraldehyde,” (H) “carbodi- imide,” and ( X ) “polyacrylamide” electrodes. The assay was per- formed in O.003M penicillin G and 0.001M phosphate buffer at a stirring rate of 390 rpm and with a standard calomel reference elec- trode. A typical penicillin fermentation period is indicated as the dashed lines (ref. 26).

FLANAGAN AND CARROLL: THIN-FILM ENZYME ELECTRODE 1097

The 90% response times varied, depending on pen- icillin concentration, between 2 and 5 min for the 25 x 15 mm electrodes and between 30 and 60 s for the 5 x 5 mm penicillinase electrodes. Figure 6 shows a typical transient response curve which is clearly bi- phasic. As a rigorous kinetic analysis of enzyme elec- trodes is complex24 and, as we are interested in the kinetics only in so far as they may allow a more rapid assay, we have adopted an arbitrary fitting procedure. We have used a nonlinear regression to fit the transient response to a double-exponential:

[V(maximum) - V(t)l/[V(maximum) - V(t = O ) ] = A[exp(-k,t)] + (1 - A)[exp(-kzt)l (6)

Figure 6 also shows the best fit to eq. (6). As the re- sponse of an enzyme electrode may, in part, be limited by a diffusion-controlled step and hence depend on a stirring rate, we have examined the transient responses and the constants obtained on fitting to eq. (6) as a function of stirring rate. Figure 7 shows that the ar- bitrary rate constant, kl , correlates well with stirring rate and, consequently, must represent a diffusion- controlled step. The absence of this step below 550 rpm in the case of the “carbodiimide” electrode and its presence at all stirring rates in the case of the “glu- taraldehyde” electrode is consistent. The latter im- mobilization technique leads to a comparatively thick membrane, while the former leads, at least in theory, to a monomolecular layer. Parameters k2 and A were, statistically, highly correlated making any definitive statements about them difficult. Parameter k2 did not vary significantly with stimng rate (mean 0.729 k 0.145). The total response, i.e., the potential change shown in Figures 2-5, did not vary by more than t 5% between

0-0 0 500 lo00

Stirring Rote, rpm

Figure 7. The rate constant, k , , as a function of stirring rate for a (A) 5 x 5 mm “glutaraldehyde” electrode and for a (H) 25 X 15 mm “carbodiimide” electrode for assays in 0.003M penicillin G , 0.001M phosphate buffer, pH 7.0, with a calomel reference electrode.

values of 100 and 800 rpm. Thus, the stirring rate pre- sents no problems if the total potential change is used to measure the penicillin concentration but the strong dependence of k , on stirring rate precludes the use of a kinetic analysis in such measurements.

CONCLUSIONS

Antimony-antimony-oxide thin-film pH electrodes can be used as the base pH transducers in enzyme electrodes. The surface dissolution of this sensor dic- tates that a crosslinked protein film, or any analogous immobilization procedure, is more appropriate than the covalent attachment techniques which would be the techniques of choice in the case of a glass support. The thin-film sensor offers several advantages over the use of more conventional pH electrodes as base trans- ducers. It has been demonstrated that it will allow the fabrication of cheap disposable multigate sensors with considerable potential for miniaturization. All these are prerequisites for the successful development of reliable enzyme electrodes suitable for commercial exploitation.

One of the authors (MTF) is the Standard Telephone and Cable Fellow in Bioelectronics and wishes to thank Standard Telephone and Cable Ltd. for permission to publish this work. The other author (NJC) wishes to thank the SERC for support throughout the period of this work.

” 0 50 100 150 200 400 600

Time, seconds

Figure 6. The experimental (-)transient response for an assay in 0.003M penicillin G and 0.001M phosphate buffer, pH 7.0, at a stimng rate of 755 rpm and the best fit of this data to (0) eq. (6) for a 25 X 15 mm “glutaraldehyde” electrode versus a calomel refer- ence electrode.

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1098 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 28, JULY 1986

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FLANAGAN AND CARROLL: THIN-FILM ENZYME ELECTRODE 1099