520 ION-SELECTIVE ELECTRODES / Enzyme Electrodes

Oehme F (1991) Liquid electrolyte sensors: potentiometry, Ross JW, Riseman JH, and Kruger JA (1973) Potentiomet-
amperometry, and conductometry. In: Göpel W, Hese J, ric gas sensing electrodes. Pure and Applied Chemistry
and Zemel JN (eds.) Sensors. A Comprehensive Survey, 36: 473–487.
vol. 2, ch. 7. Weinheim: VCH. Veselý J and Štulı́k K (1987) Potentiometry with ion-
Riley M (1979) Gas-sensing probes. In: Covington AK (ed.) selective electrodes. In: Kalvoda R (ed.) Electroanalytical
Ion-selective Electrode Methodology, vol. 2, ch. 1. Boca Methods in Chemical and Environmental Analysis, ch. 6.
Raton, FL: CRC Press. New York: Plenum.

Enzyme Electrodes
M Mascini and I Palchetti, Università degli Studi di of sensors based on field-effect transistor (so called
Firenze, Firenze, Italy ion-selective field-effect transistor, ISFET).
& 2005, Elsevier Ltd. All Rights Reserved. Nowadays, ion-selective sensors are constructed
using various advanced methods such as thick- and
thin-film technology. A thick-film sensor comprises
Introduction layers of special pastes (thickness 10–50 mm) deposit-
ed onto an insulating substrate; the method of film
Potentiometric enzyme electrodes form a large group
deposition is the screen-printing technique. Thin-film
of catalytic biosensors, resulting from a thin layer of
electrochemical transducers are built up by the suc-
enzyme integrated within or intimately associated
cessive deposition and patterning of dielectric and
with a potentiometric transducer. The enzyme gene-
conductive materials, on top of an optically flat and
rates a reaction that enables determination of the
polished substrate. The deposition of the thin metallic
substrate (the target analyte), providing a highly
films (thickness 10–200 nm) is generally carried out
selective and sensitive method for the determination
by classical evaporation or sputtering of a solid metal
of a given, ionic or nonionic, substrate. The deter-
source; then, photolithography and other techniques
mination of the electrode potential gives a direct in-
can be envisaged to pattern the electrode material.
dication of the concentration of the analyte. The
Using these technologies, microfabricated arrays of
signal obtained is proportional to the logarithm of
sensors and biosensors have been developed.
the concentration, following the Nernst law.
Interesting features of potentiometric transducers
are simplicity of instrumentation (only a potentio-
meter is needed), low cost, selectivity, and the non-
General Features of Enzyme
destructive nature of the analytical procedure.
Electrodes
Potentiometric Transducers Biosensor Assembly

Guilbault and Montalvo were the first, in 1969, to In order to prepare the biosensor, the enzyme has to be
detail a potentiometric enzyme electrode. They de- immobilized in the form of a thin layer at the surface
scribed a urea biosensor based on urease immobi- of the transducer. Among the several methods for im-
lized at an ammonium-selective liquid membrane mobilization available, the following are the most
electrode. Since then, over hundreds of different ap- generally employed: (1) physical entrapment within
plications have appeared in the literature, due to the an inert polymeric membrane (in this case the enzyme
significant development of ion-selective electrodes is mixed with a monomer solution, which is then poly-
(ISEs) observed during the last 30 years. The elec- merized to a gel – polyacrylamide gel, starch, agar
trodes used to assemble a potentiometric biosensor gel, etc., thus trapping the enzyme) or behind a
include glass electrodes for the measurement of pH membrane (in this case the enzyme solution is simply
or monovalent ions, ISEs sensitive to anions or cat- confined by an analyte permeable membrane, such as
ions, gas electrodes such as the CO2 or the NH3 a dialysis membrane, as a thin film covering the in-
probes, and metal electrodes able to detect redox dicator electrode; this method is also called micro-
species; some of these electrodes useful in the con- encapsulation); (2) covalent bonding of the enzymes
struction of potentiometric enzyme electrodes are on membranes or surfaces activated by means of bi-
listed in Table 1. functional groups or spacers, such as gluteraldehyde,
In the 1980s, ion-selective membranes were used carbodiimide (when the enzyme is bound to the solid
for the first time as the molecular recognition element support using a bi- or multifunctional agent, such as
 ION-SELECTIVE ELECTRODES / Enzyme Electrodes 521

Table 1 Potentiometric enzyme electrodes

Electrochemical sensor Enzyme Metabolite Measurement range (mmol l
1)

Glass, pH Urease Urea 0.05–5

Glucose oxidase Glucose 0.1–1
Penicillinase Penicillin 0.01–3
Acetylcholinesterase Acetylcholine 0.01–10
Antimony, pH Urease Urea 0.1–10
ISFET, pH Urease Urea 1–1000
Glucose oxidase Glucose 1–50
Penicillinase Penicillin 1–20
NH3 probe Urease Urea 0.05–50
Glutaminase Glutamine 0.05–5
Phenylalanine ammonia lyase Phenylalanine 0.05–1
Asparaginase Asparagine 0.1–10
AMP deaminase 50 -AMP 0.1–10
Phosphodiesterase þ AMP deaminase 30 ,50 -cyclic-AMP 0.01–10
Creatinine deaminase Creatinine 0.1–10
CO2 probe Urease Urea 0.1–10
Uricase Uric acid 0.1–2.5
Decarboxylating enzymes Tyrosine 0.1–2.5
Asparagine 0.1–10
Lysine 0.1–30
Glutamate 0.5–5
Ammonium (neutral carrier based) Urease Urea 0.01–10
Creatininase Creatinine 0.01–5
Asparaginase Asparagine 0.01–1
Iodide Glucose oxidase Glucose 0.01–10
Amino acid oxidase Amino acid 0.01–10
Cholesterol oxidase Cholesterol 0.01–1
Redox Laccate deydrogenase Lactate 0.1–1
Cyanide b-Glucosidase Amygdalin 0.1–100

gluteraldehyde, which forms a bridge between electrode; (2) diffusion of the substrate through the
biocatalytic species or proteins, the technique is membrane to the enzyme-active site; (3) a reaction
also called cross-linking or reticulation); and (3) im- occurring at the active site; (4) product formation in
mobilization on commercially available activated the enzymatic reaction and its transport through the
membranes such as Immunodyne produced by membrane to the surface of the electrode; and (5)
Pall Industries, or Ultra-Bind produced by Gelman measurement of the product at the electrode surface.
Sciences. The first step, transport of the substrate, is most
Several enzymes can be immobilized within the critically dependent on the stirring rate of the solu-
same reaction layer, in order to increase the range of tion, so that rapid stirring will bring the substrate
possible biosensor analytes, provide efficient regene- very rapidly to the electrode surface. If the membrane
ration of enzyme cosubstrates, or to improve the is kept very thin using highly active enzymes, then
biosensor selectivity by decreasing the local concen- steps (2) and (4) are eliminated or minimized; since
tration of electrochemical interfering substances. step (3) is very fast, the response of an enzyme elec-
Generally, the technique of chemical immobiliza- trode should theoretically approach the response
tion (covalent bonding) is preferred. This procedure time of the base sensor. Many researchers have
improves the enzyme stability and defines a diffusion shown experimentally that one can approach this
layer where the product, formed by the catalytic ac- behavior using a thin membrane and rapid stirring.
tion of the enzyme, diffuses partly to the electrode
surface and partly back to the bulk of the solution;
therefore, by defining the steady state one obtains Analytical Features of Enzyme
a reproducible potential value. However, the real Electrodes
advantage of the enzyme immobilization through
Concentration Range
chemical bonding is the lifetime of the final probe,
which can reach 6–12 months. The maximum concentration of substrate that can
An enzyme electrode operates via a five-step pro- be measured with an enzyme electrode is governed
cess: (1) transport of the substrate to the surface of the by Km (the Michaelis–Menten constant). If the
522 ION-SELECTIVE ELECTRODES / Enzyme Electrodes

Table 2 Km values reported for soluble enzymes and maximum concentration values of the linear concentration range reported for
each biosensor probe

Enzyme Substrate Km (mmol l
1) Concentration value (mmol l
1)

Urease Urea 10 10
Glucose oxidase Glucose 7.7 35
L-Amino acid oxidase Leucine 1.0 10
Invertase Sucrose 0.45 1
Alkaline phosphatase p-Nitrophenylphosphate 0.1 1
Lactate oxidase Lactate 0.7 20
Pyruvate oxidase Pyruvate 1.7 2
Creatininase Creatinine 0.33 1
Acetylcholinesterase Acetylcholine 0.09 1
Uricase Uric acid 0.017 1

concentration is near to Km, the linear relation be- 180

Units urease per cm3 gel
* *
tween substrate concentration and the extent of en- * 3.0
zyme reaction fails and a limiting value is reached. * 52.5
However, the picture is not always as straightfor- * 65.6
120 * 87.4
ward as this. Table 2 lists the maximum concentra-
E (mV)
*
tion values of substrates measured with enzyme
probes and compares them with Km data for soluble
enzymes. In general, the maximum concentration of 60 *
*
a substrate exceeds the Km value by approximately *
one order of magnitude.
The difficulty of measuring high concentration is 0
usually overcome by diluting the sample. This pro- 1 2 3 4 5
cedure has the advantage of the control of pH and –log urea (mol l–1)
ionic strength by the use of suitable buffers, so that
Figure 1 Calibration of a potentiometric urease electrode for
samples and standards are measured under identical urea. Curves are related to different loadings.
conditions.
Immobilization of the enzyme is thought to in- The amount of enzyme to be used depends on the
crease the Km value, the increase being related to immobilization procedure and on the enzyme purity.
change of substrate/carrier, diffusion effects, or As a rule of thumb, B10–20 IU per membrane is
changes in enzyme structure. generally sufficient to give an excellent response. For
The minimum concentration is often related to the the less stable, soluble, and physically entrapped en-
minimum amount of electroactive substance that can zyme electrodes, an excess of enzyme should be used,
be measured with the electrochemical probe. Gener- that is, 50 IU cm
2 of electrode surface, so that a loss
ally, with ISE or gas potentiometric probes the limit of enzyme does not affect the observed responses.
of concentration measurable is 10
5 mol l
1. The Likewise, purified enzymes should be used so as to
calibration graphs are therefore often S-shaped, promote fast response rates.
leveling off at high concentration due to the Km or
Km-related maximum concentration measurable and Response Time
at low values by the performances of the potentio-
metric base probe (Figure 1). Mathematical models describing response times have
One of the most important parameters concerning been considered in the literature. This is the essential
the concentration range is the activity of the enzyme feature of an assembled probe, and depends on the
in the membrane layer. As a general rule, the slope of procedure of assembling the probe, specifically on
the sensor will increase with increasing amount of the method of immobilization, the electrochemical
enzyme up to a limiting value. The activity of the probe employed, the geometry of the measuring cell,
enzyme determines the percentage of reaction of the and the kinetics of the enzymatic reaction.
substrate, and hence the amount of electroactive
Diffusion of Substrate
species. However, with increasing amounts of en-
zyme loading, the percentage of substrate reacting The difference in response between an unstirred so-
reaches a limit that matches its complete transfor- lution and a stirred one can decrease the response
mation. time from 10 to 1–2 min or less. Also, the potential is
 ION-SELECTIVE ELECTRODES / Enzyme Electrodes 523

a function of stirring rate related to the amount of 49 in order to obtain complete conversion of NH4þ
substance brought to the electrode surface and to its into NH3, and to obtain a shorter response time of
reactivity. Hence, for fast response times and steady the electrochemical probe. However, few enzymes
states, a fast but constant rate of stirring of the can function at such high pH, so a compromise is
substrate solution is recommended. necessary. With the carbon dioxide sensor, the sam-
ples should be kept at pH o5 to obtain a complete
Substrate Concentration conversion to carbon dioxide of hydrogencarbonate;
The rate of enzymatic reaction increases with sub- however, these low pH values often interfere with the
strate concentration; for example, in the case of a enzyme reaction, and a compromise is necessary be-
cyanide electrode coupled with immobilized b-gluco- tween optimum pH for enzyme activity and optimum
sidase enzyme, a response time of 20 s for 10
1 pH range of response of the probe.
mol l
1 amygdalin and 1 min for 10
4 mol l
1
Temperature Effect
amygdalin is obtained. Rather than waiting until an
equilibrium potential is reached, the rate of potential The effect of increasing temperature is twofold: an
change ðDE=DtÞ can be measured, the result being increase in the rate of reaction gives faster response
proportional to substrate concentration. time and a shift in the equilibrium value due to
variations in the equilibrium constant. Electrochemi-
Enzyme Concentration cal sensors of the gas-permeable membrane type
There is a twofold effect on increasing the enzyme (ammonia and carbon dioxide) lead to an additional
activity in the layer that is in close proximity to the effect, since the gas membrane and the features of the
electrode surface. First, a complete conversion of diffusion are sensitive to temperature variations. De-
the substrate into products will be ensured; second, spite the above considerations, a classical bell-shaped
the response time of the electrode will be affec- curve is almost always obtained when recording the
ted. The increase in the amount of enzyme affects the response of the probe as function of temperature.
thickness of the membrane. This results in an in- Room temperature, or 251C (controlled to 70.21C is
crease in the time required for the substrate to diffuse recommended), is often employed when using an
through the membrane. Hence, for the best results, it electrochemical probe, although when using a gas
is recommended that an enzyme of as high an activity permeable membrane control to 70.11C is required.
as possible is used in order to ensure rapid kinetics by
Thickness of the Enzyme Membrane
achieving the thinnest possible membrane.
The time required to reach steady-state potential
pH Effect reading is dependent on the enzyme layer thickness
Every enzyme has an optimum pH at which it is most because of the diffusion parameter for the substrate
active and a certain pH range within which it does to reach the active sites of the enzyme and of the
not exhibit reactivity. The pH ranges of immobilized electroactive species to diffuse through the mem-
enzymes are different from those of soluble enzymes brane to the sensor. A mathematical model relating
because of the microenvironment. Highly negatively the thickness of the membrane, d, the diffusion co-
charged carriers create a lower pH at the boundary efficient, D, the Michaelis constant, Km, and the
layer between the carrier and the bulk solution. maximum velocity of the enzyme reaction, Vmax, has
Thus, the enzyme is in a more acidic environment been developed:
than the bulk solution. The reverse occurs with
Vmax d2 =DKm ¼ V
positively charged supports. Even for uncharged
supports, the overall charges of the enzyme may be
sufficient to cause changes in the apparent pH where V compares the rate of chemical reaction in
optimum. However, in general, the greater the charge the membrane with the rate of diffusion through the
on the support, the greater is the effect, particularly membrane. Larger V indicates faster enzyme cataly-
with charged substrates. The optimum pH should be sis relative to the diffusion process.
used for obtaining the fastest response, but this is not Membranes as thin as possible are recommended
always possible because the electrode sensor may not for the best results, and this is helped by the use of a
itself give the optimum response at the pH of the highly active enzyme.
enzyme reaction. Therefore, a compromise is usually
Effect of Additional Membranes
necessary. This is especially important for the am-
monia and carbon dioxide electrodes. For ammonia Besides the enzymatic reacting layer, many biosen-
electrochemical sensors, samples should have a pH sors, especially designed for biological or clinical
524 ION-SELECTIVE ELECTRODES / Enzyme Electrodes

applications, incorporate one or more membranes, Interference Effects

which serve mainly two important functions:
For each enzyme-based sensor interferences fall into
(1) Protective barrier: to prevent the interference of
two categories, namely interference of the electrochem-
molecules, such as proteins or cells of biological
ical probe and interference with the enzyme reaction.
samples, with the reaction layer or to decrease the
influence of electrochemical species detected by the Interference of the Electrochemical Probe
transducer. It also reduces leakage of the reacting
layer components into the sample solution. (2) Dif- In the development of a urea electrode, the first ap-
fusional outer barrier for the substrate: the linear proach was the use of a cation glass electrode, but the
dynamic ranges may be large if the sensor response is resulting probe could not be used for assaying urea in
controlled by the substrate diffusion through the blood and urine because the sensor responded to so-
membrane and not by the enzyme kinetics. This is dium and potassium ions.
achieved by placing a thin outer membrane over a The sensor was improved by using the non-
highly active enzyme layer. actin-based ammonium ISE. This ionophore has
Generally, the thinner the membranes, the shorter selectivity constants KNH4,K of 0.15 and KNH4,Na of
is the biosensor response time. 1.3  10
3, thus partially eliminating the response of
these ions by the sensor. Several articles have been
published on this principle, taking into account the
Electrode Probe residual effect of sodium and potassium.
A limiting factor on the speed of response of an en- By using an ammonia electrode with a gas-perme-
zyme-based electrode is the response time of the base able membrane that effectively excludes ions, it is
electrode. Electrodes based on gas-permeable mem- possible to obtain the desired selectivity. Several
branes (ammonia or carbon dioxide) have longer re- assemblies have been studied to obtain rapid and
sponse times than those without such membranes reliable sensors.
(ISEs, pH glass electrodes, ISFETs). In both cases, the The carbon dioxide probe can be used as a trans-
response time of the electrode is the limiting factor at ducer for urea (Table 1); it is selective because the
low substrate concentration. Response times of the gas-permeable membrane excludes ions and other
order of 10–30 s can easily be obtained for concen- chemicals. However, many samples, especially in
trations in the range 0.1–1 mmol l
l. biological or clinical chemistry, have large levels of
bicarbonate, which will interfere with this probe.
ISEs display different problems of interference when
Washing Time of Probe used as enzyme-based probes. In the cyanide sensor for
Because of a build-up of product in the enzyme amygdalin, sulfide can interfere, while an iodide elec-
membrane, enzyme electrodes require washing prior trode used for measuring glucose is susceptible to the
to contact with the next sample. The washing time presence of thiocyanate, sulfide, cyanide, and silver
varies from just 20 s for urease in conjunction with ions. Moreover, oxidizable compounds present in
an ammonia electrode to as long as 10 min for urease blood (e.g., uric acid, tyrosine, ascorbic acid, and
with a pH electrode. The washing time increases iron(II)) compete for hydrogen peroxide. These com-
with enzyme membrane thickness, as also observed pounds need to be removed by sample pretreatment.
for additional membranes discussed above. It will Finally, with regard to pH electrodes coupled to en-
also depend on the enzyme used and on the charac- zymes (Table 1), any acidic or basic component will
teristic of the base sensor itself, and will be affected interfere, as will a variable buffer capacity as found in
by diffusion and kinetic effects. The use of flow in- clinical and biological samples. However, by adjusting
jection analysis simplifies the procedures, since the the pH before initiating the enzyme reaction, and assu-
carrier stream serves to wash out between samples. ming that only the enzyme reaction will give a pH
Deterioration of the enzyme electrode can be seen change, the effects can be minimized.
in three changes in the response characteristics:
Interferences with the Enzyme Reaction
(1) with age the upper limit will decrease from
B10
1 to 10
2 mol l
1; (2) the slope of the cali- These fall into two classes, namely those where other
bration curve of potential versus log(concentration), substrates are present in the sample and those
59 mV per decade, Nernstian, will drop to 50, 40, or involving base enzyme activation or inhibition. With
perhaps 30 mV per decade or lower; (3) the response some enzymes, such as urease, the only substrate that
time of the electrode, originally 30 s to 2 min (ap- reacts at a reasonable rate is urea; hence, a urease
proximately the same as that of the base sensor), will electrode is almost specific for urea. Likewise,
become longer as the enzyme ages. uricase is almost specific for uric acid.
 ION-SELECTIVE ELECTRODES / Enzyme Electrodes 525

Others, such as penicillinase, will promote the re- nonactin, a neutral carrier, as the base sensor, together
action of many substrates. Thus, ampicillin, naficillin, with immobilized urease in a polyacrylic gel was used.
penicillin G, penicillin V, cyclibillin, and dicloxacillin
Creatinine
can be determined with a penicillinase electrode.
D- and L-amino acid oxidases are even less selective. Creatinine is another diagnostic indicator of kidney
The former, when coupled to an electrode, responds function. Creatinine can be measured immobilizing
to D-phenylalanine, D-alanine, D-valine, D-methionine, purified creatininase (reaction [II]) onto nylon net
D-leucine, D-norleucine, and D-isoleucine, while the and coupling it with an ammonia gas electrode:
latter responds to L-leucine, L-tyrosine L-phenylala-

! NH3 þ creatine
Creatininase
nine, L-tryptophan, and methionine. Creatinine ½II
For samples containing several substrates, a prelimi- The residual ammonia of blood serum can be
nary separation should be considered or the total must removed using an enzyme reactor system, consisting
be determined. In the case of L-amino acid assay the of soluble NADPH and a-ketoglutarate, with gluta-
use of decarboxylating enzymes acting selectively on mate dehydrogenase immobilized on nylon tubes; this
different amino acids is an attractive possibility, and system is effective in removing 98% of the ammonia
enzyme electrodes of this type are known for L-tyro- present in human blood and urine samples in 50 s. The
sine, L-phenylalanine, and L-tryptophan. These sensors entire process was carried out in a single flow stream.
will be coupled with a carbon dioxide base sensor.
Enzyme inhibitors also need to be taken into ac- Glucose and Sugar Electrodes
count. These include heavy metal ions, such as silver, An iodide-selective electrode can be used to measure
mercury, and sulfydryl-reacting organic compounds, glucose concentration. The measurement is based on
such as p-chloromercuribenzoate (due to their reac- reactions catalyzed by glucose oxidase (GOx) (reac-
tion with the free S–H groups at the active site of tion [III]) and peroxidase (POx) (reaction [IV]):
many enzymes, especially the oxidases).

! gluconic acid þ H2 O2
GOx
Glucose þ O2 ½III
Examples of Enzyme Electrodes þ POx
H2 O2 þ 2I
þ 2H
! 2H2 O þ I2 ½IV
Based on ISEs
The highly sensitive iodide sensor monitors the de-
Table 1 lists enzyme electrodes that have been pre-
crease in the iodide activity at the electrode surface.
pared for analysis of common substrates together
The assay of glucose was performed both in a stream
with the enzyme used, the sensor, and the range of
and at a stationary electrode. Pretreatment of the blood
concentrations determinable. The most important
sample was required to remove interfering reducing
features of some of these are described here.
agents such as ascorbic acid, tyrosine, and uric acid.
Urea Moreover, pH electrodes can be used to detect the
gluconic acid formed by the GOx reaction.
Urea is a diagnostic indicator for kidney function.
Urea can be potentiometrically determined following Amino Acids
the enzymatic reaction of the enzyme urease (reac- Enzyme electrodes have been widely used for the assay
tion [I]) coupled with a variety of potentiometric of amino acids in clinical analysis since several amino
transducers such as the pCO2 electrode, the pNH3 acids (tyrosine, phenylalanine, tryptophan, methio-
electrode, the pH electrode, and the pNH4 electrode: nine) are important diagnostic health indicators.
The transducer can be an ammonium ISE ionophore
H2 N2CO2NH2 þ H2 O
! CO2 þ 2NH3
Urease
½I (nonactin-based), a gas sensor for ammonia or carbon
dioxide, and an iodide-selective sensor. The latter can
The products of the enzymatic reaction given above be coupled with the enzymes L-aminooxidase (L-AOx)
also dissociate: and peroxidase (e.g., coimmobilized in a polyacryl-
amide gel), which catalyses reactions [V] and [VI]:
CO2 þ H2 O2HCO

3 þH
þ

NH3 þ H2 O2NHþ
4 þ OH
! H2 O2 þ other products
L-AOx
L-Phenylalanine ½V
The first urea electrode was prepared in 1969 by
þ POx
H2 O2 þ 2I þ 2H
! 2H2 O þ I2 ½VI
immobilizing urease in a polyacrylamide matrix on
nylon or Dacron nets. These nets were then placed Specific sensors for phenylalanine, tyrosine, gluta-
onto a glass cation-selective electrode. To improve the mine, lysine, and methionine have been described in
selectivity, an ammonium-ion selective electrode with the literature.
526 ION-SELECTIVE ELECTRODES / Clinical Applications

Penicillin See also: Clinical Analysis: Glucose. Enzymes: Immo-

bilized Enzymes. Ion-Selective Electrodes: Overview.
Penicillin is determined by cleaving the amide bond Liquid Chromatography: Amino Acids.
of the b-lactame ring with penicillase to produce
penicilloic acid. This acid is detectable by a pH elec-
Further Reading
trode covered with a thin film of penicillase and the
biosensor produced was used to monitor penicillin Cass AEG (ed.) (1990) Biosensors. A Practical Approach.
concentration in many different fermentation broths. Oxford: Oxford University Press.
Eggins BR (2002) Chemical Sensors and Biosensors.
Neurotransmitters London: Wiley.
Guilbault GG (1984) Analytical Uses of Immobilized
Neurotransmitters may be determined using the re- Enzymes. New York: Dekker.
spective hydrolases. Acetylcholine is hydrolyzed by Guilbault GG and Mascini M (eds.) (1987) Analytical Uses
acetylcholinesterase (AChE) according to reaction of Immobilized Biological Compounds for Detection.
[VII]: Medical and Industrial Uses. Dordrecht: Riedel.
AChE Mascini M and Palleschi G (1989) Design and applications
Acetylcholine
! acetate þ Hþ þ choline ½VII of enzyme electrode probes. Selective Electrode Reviews
11: 191.
which can be monitored by immobilizing AChE on a Mosbach K (ed.) (1988) Immobilized enzymes and cells,
pH electrode. Part D. In: Methods in Enzymology, vol. 137. London:
Academic Press.
Inhibitor-Sensitive Potentiometric Enzyme Ramsay G (ed.) (1998) Commercial Biosensors, Applica-
Electrodes tion to Clinical, Bioprocess, and Environmental Samples.
New York: Wiley.
Some examples of potentiometric enzyme electrodes Schmid RD and Scheller F (eds.) (1989) Biosensors. Applica-
have been developed for indirect monitoring of tion in Medicine, Environmental Protection and Process
organic pesticides or inorganic (heavy metals, fluo- Control. Weinheim: VCH.
ride, cyanide, etc.) substances that reduce the activity Thevenot DR, Toth K, Durst RA, and Wilson GS (1999)
of the immobilized enzyme on the transducer. Para- Electrochemical biosensors: recommended definitions
meters such as enzyme concentration, substrate con- and classifications. Pure and Applied Chemistry 71(12):
centration, pH, incubation time can affect the 2333–2348.
biosensor response, and have to be optimized. A Tran Minh C (1993) Biosensor. Paris: Chapman and Hall
and Masson.
classical example is the monitoring of organophos-
Turner APF (ed.) (1992) Advances in Biosensors, vols. l
phorous and carbammate pesticides (whose mecha- and 2. London: JAI Press.
nism of action is the inhibition of AChE, the enzyme Turner APF, Karube I, and Wilson G (1986) Biosensors,
implicated in the neurotransmission) using a glass Fundamentals and Applications. Oxford: Oxford
electrode with immobilized AChE. Science.
Heavy metals and fluoride ions have been reported Wise DL (ed.) (1990) Bioinstrumentation Research,
to inhibit the activity of the enzyme urease. Development and Applications. London: Butterworths.

Clinical Applications
U E Spichiger-Keller, Centre for Chemical Sensors and the recognition process. Fiber optical sensors have
Chemical Information Technology, Zurich, Switzerland been developed in response to the need for selective,
& 2005, Elsevier Ltd. All Rights Reserved. reliable sensors for medical use, especially for pa-
tient-near offline or continuous in vivo monitoring,
This article is reproduced from the first edition, pp. 2341–2353,
& 1995, Elsevier Ltd., with revisions made by the Editor. related to the problems inherent in electrochemical
methods. Also, optical test kits for blood ions
are commercially available. However, electrodes of-
fer a broad field of applications in patient-near-moni-
Introduction toring as well as in large automated biochemistry
An ion-selective electrode (ISE) is a chemical sensor analyzers.
that can quantitatively and reversibly measure an ion Ion-selective electrodes introduced in chemical
activity based on a potentiometric transduction of instruments are most frequently based on liquid