Response of ammonium-selective microelectrodes based on the neutral carrier nonactin

Talanta,Vol. 35, No. 9, pp. 728-730, 1988 Copyright 0

Printed in Great Britain.All rights reserved

0039-9140/88 $3.00 + 0.00 1988 Pergamon Press plc

RESPONSE OF AMMONIUM-SELECTIVE MICROELECTRODES BASED ON THE NEUTRAL CARRIER NONACTIN D. DE BEER and J. C. VAN DEN HEUVEL Department of Chemical Engineering and Biotechnological Centre, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands (Received 10 December

1987. Revised 19 April 1988. Accepted 12 May 1988)

Sunmmry-Ammonium-selective microelectrodes made with an ion-exchanger based on nonactin and having a tip diameter of 1 pm have been developed. The response of the electrode is linear from 10m5to lo-‘M NH: and the response time is 1 min. Applications of these electrodes can be found in biotechnology and microbial ecology, but the low selectivity with respect to K+ and Na+ limits their use to low salt environments. Concentration gradients in gels containing cross-linked urease were measured and found to be in accord with macrokinetic measurements.

EXPERIMENTAL

Microelectrodes are unique tools for the measurement of concentrations in extremely small volumes, and of concentration gradients over pm distances. They were first used for intracellular analysis

Two types of liquid ion-exchanging - - membrane (LIX) were use&type A was a saturated solution of NH:-selective ligand (75% nonactin + 25% monactin, Fluka AG) in tris(ethylhexy1) phosphate (TEHP), and type B was a solution oflO% the same ligand and 1% oftktraphenylborate (tPB) in o-nitroohenvl n-octvl ether (NPOE). Glass cauillaries 112mm in diameter (Clark GC12bF-15) were drawn with an automatic horizontal puller (Anna puller, Biological Centre Amsterdam) into micropipettes. During the pulling procedure air was blown at 1 ml/set along the capillaries to reduce the ratio of the outer and inner diameters of the tip of the micropipettes. The outer tip diameter of the micropipettes was about 1 pm, and their resistance when filled with 3M potassium chloride was 5 mR. Immediately after being pulled, the tips were silaned by dipping them in a 20% solution of trimethylchlorosilane in carbon tetrachloride for 15 set, followed by baking at 130” for 15 min. The electrode tips were filled with sensor through capillary attraction by dipping the tips in the sensor at 40”. The electrode shafts were subsequently filled with 0.03M potassium chloride as electrolyte solution by means of a small tube. Air bubbles were removed by heating the electrodes locally with a heating loop. The cell used for calibration was

by animal physiologists and neurophysiologists. Nowadays their application has reached the fields of microbial ecology and biotechnology, for use in microenviromnental studies. Interesting results have been obtained for sediments and microbial films with pH and oxygen microelectrodes.’ Besides their complicated construction, oxygen microelectrodes have the disadvantage of disturbing the oxygen distribution patterns because of their size of 5 pm and their consumption of oxygen.’ Until now, no other microelectrodes have been available to measure the substrates relevant in this context.

of

In order to study kinetics and mass transfer in bacterial aggregates, microelectrodes have been constructed with an ammonium-selective liquid membrane. The electrodes have a tip diameter of about 1 pm and are relatively simple to prepare. TO test the

reliability of these microelectrodes under experimental conditions, product gradients have been measured in a well-defined model system. For this purpose, spherical gel beads were prepared containing immobilized urease, which catalyses the hydrolysis of urea to ammonium and carbonate. The product gradients were compared with data obtained from macrokinetic measurements. Urease-containing gels may be employed for the treatment of urea containing waste water from fertilizer industries etc.3 728

The electrode assembly was placed inside a Faraday cage. Measurements were made with a continuous-flow cell at 20 f 0.5”. The solutions inside and outside the Faraday cage were electrically separated by drip chambers. The potential difference between the Ag/AgCl wires was transmitted through a triaxial input cable (Keithley 6011) to an electrometer (Keithley 617) placed outside the Faraday cage. Selectivity factors were determined by the separate solution method!.’ The urease-containing gels were prepared as follows. Urease was cross-linked with bovine serum albumin.6 A 0.75% suspension of the cross-linked enzyme in demineralized water containing 0.001% Triton X-100 was pre-

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pared by sonification (2 x 30 sec. 8 W/ml). One part of the suspension was mixed with four parts of a 2.5% agar solution at 40”. The enzyme-agar suspension was dripped into oil at 10” to form spherical gel beads with a diameter of 3.2mm. The homogeneous distribution of the enzyme was verified calorimetrically with Coomassie Brilliant Blue. The gels were kept at pH 9.0 in a buffer consisting of 0.02M triethylethylenediamine (TEEDA) and O.OOlM ethylenediaminetetra-acetic acid (EDTA). The gel beads were fixed in the flow cell with two entomological-specimen needles. Electrodes were positioned in the gels by a micromanipulator driven by a stepper motor. During concentration measurements on the gel, buffer supplied with the desired concentration of urea was pumped through the flow cell. Before each experiment the gels were incubated in this buffer for 40 min to achieve a steady state. The maximum reaction rate (I’,,) and Michaelis constant (K,,,) of the cross-linked enzyme were determined by measuring the rate of ammonium carbonate production with an ammonium-selective macroelectrode.’ The apparent kinetic parameters of the immobilized enzyme in the gel beads were measured in the same way. When the conversion in a homogeneous gel bead is reaction-limited, it can be deduced that the product concentration gradient in the gel near the interface with the bulk liquid is given by

L-1 dc

I’,,, SR dr ,xR= 3(S + K,)D

729

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(1)

where c is the product concentration, S the substrate concentration, R the radius of the gel bead, D the diffusion coefficient, and r the distance from the centre. The nature of the limitation was determined by calculating the modified Thiele modulus Gp.s If 0, is < 1 the conversion is not limited by diffusion.

RESULTS

The responses of both types of microelectrodes to concentrations of 10-S-lO-iM ammonium carbonate

in demineralized water were linear, with slopes of 50-55 mV/log[NH:] (Fig. 1). The resistance of the electrodes with LIX type A depended on the NH: concentration and varied from 20GRat lo-‘MNH: to90GRat 10-SMNH$.The 90% response time (tW) was about 10 sec. The resistances of the electrodes with LIX type B ranged from 10 to 40 GQ and were independent of the NH: concentration. The response time (tw) was 60 sec. The drift of the signal after a stabilization period of about 0.5 hr was l-2 mV/hr. The response of the electrode was independent of pH in the range from 5 to 8 but at higher pH values the response decreased because of the shift in the ammonia/ammonium equilibrium.

2&O-

E (mV)

200.

-LOi 1

2

3

i

0.12 0.002 0.00017

6 Cl1

Fig. 1. Calibration curves of microelectrodes with LIX type B. (0) demineralized water, (A) pH 9.0 buffer, 0.02M TEEDA and O.OOlM EDTA.

In a 0.02M TEEDA buffer at pH 9.0 the lower limit of linear response increased to 10-4M NH: The selectivity factors of the different types of electrode are given in Table 1. Electrodes with NPOE as solvent (type B) for the ion-exchanger showed a higher selectivity than those with TEHP (type A). Addition of 1% tPB did not influence the selectivity with respect to Na+ and K+, but increased that with respect to Ca2+. The electrodes could be used for 12 hr, during which the detection limit and the slope factor remained constant. However, after 24 hr the detection limit had sometimes increased by a factor of 10. As a consequence, freshly prepared electrodes were used for each experiment. Product gradients in gel beads containing crosslinked urease were measured with electrodes of type B. They were almost symmetrical, and their size depended on the substrate concentration, as shown in Fig. 2. The results of four experiments are summarized in Table 2. The measured slopes show good agreement with those calculated by means of equation (1) from macrokinetic measurements. In the buffer used, the K,,, value for the cross-linked enzyme, and also the apparent K,,, for the gel beads, was 0.0058M. In all experiments the modified Thiele

Microelectrode

Kf Na+ Ca2+

5 -logINH,

Table 1. Selectivity factors KNH,,, for microelectrodes with different types of liquid membranes and a macroelectrode

Macroelectrode’ LIX type A

L

LIX type B LIX type A

(no tPB)

0.85 0.67 0.07

0.4 0.02 0.07

(with tPB) 0.38 0.02 0.002

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Table 2. Product concentration gradient (dc/dr) at r = R in different conditions Parameter 5 x 10-7

5, M @P

I .68 0.1 0.053

8.8 x IO-8 1.64 0.1 0.015

3 x IO-’ 1.68 0.004 0.165

6.8 x 10-n 1.64 0.004 0.055

dc/dr*, mole.l-‘.m-’ dc/drt, mole.l-‘.m-’

0.332 0.340

0.057 0.051

0.086 0.080

0.019 0.018

V_rr mole.l-‘.sec-’ R, mm

*Calculated from macrokinetic data by equation (1). t Measured with microelectrodes.

0.5

g

0.4

E 7

0.3

s <

0.2

z =

0.1

0.0 -0.002

-0.001

0

0.001

0.f

2

r(m) Fig. 2. Measured product concentration gradients in agar gels containing immobilized urease. Gels were incubated in (0) O.lM and (A) 0.004M urea.

modulus 0, was less than 1 so the reaction rate was not limited by diffusion, and equation (1) holds.

salt solutions no concentration changes of interfering ions should occur. Measurements can be made in media such as freshwater sediments, nitrifying bacterial granules9 and gels with immobilized enzymes, since in these systems no concentration gradients or concentration changes of Na+ and K+ are to be expected. In our experiments the electrode proved to be reliable, since the measured gradients corresponded quite well with macrokinetic data. In biotechnology much effort is directed to the development of mathematical models to quantify the conversion inside a biofilm or a catalyst particle containing immobilized enzymes.“’ The value of these models depends heavily on the assumed structure of the biological system, i.e., the distribution of organisms and kinetic parameters. With microelectrodes it is possible to obtain direct information from measurements inside heterogeneous biological systems. Therefore, it is to be expected that the electrode developed may have wider applications in microbial ecology and biotechnology.

DISCUSSION The electrode developed is a modification of the macroelcctrode described by Scholer and Simon.’

Simple miniaturization, however, was unsatisfactory because the resulting microelectrodes showed poor selectivity (Table 1) and often shed their membranes. Both selectivity and stability could be improved by the use of NPOE (type B) instead of TEHP (type A) as the membrane solvent, although the response time was longer. Furthermore, addition of tPB increased the selectivity with respect to bivalent cations and resulted in a resistance that was independent of the ammonium ion concentration. The selectivity of the microelectrodes with LIX type B was sufficient for our purposes, although lower than the selectivity of macroelectrodes.’ It should be noted that miniaturization increases the role of shunt resistances between the glass-membrane interface and the glass wall, generally leading to a decrease of selectivity.5 The measuring range of the electrodes for ammonium ions is limited by the presence of interfering ions. As a rule of thumb the detection limit for NH: is O.l[Na+] or lO[K+]. The electrode is therefore not suitable for physiological experiments or use in marine environments. During measurements in dilute

authors wish to thank Dr. J. Siegenbeek van Heukelom (Department of Zoology, University of Amsterdam) for technical facilities and expert advice. Acknowledgement-The

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