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Electrochemistry and Chemical Sensors: Prasad V.A. Pamidi

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Riskullah Makmur

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17

Electrochemistry and Chemical Sensors*

Prasad V.A. Pamidia

ABSTRACT
Background creatinine). Recent advances in miniaturization of enabling
Chemical sensors utilizing electrochemical and optical detec- technologies and the demand for direct whole blood mea-
tion methods have become routine analytical tools in clinical surements have transformed these sensors as ideal measure-
chemistry applications, especially for measurement of critical ment technologies for point-of-care testing, near patient
care analytes (blood gases, electrolytes, metabolites) directly testing, or at-home monitoring. Affinity sensors utilizing
in whole blood at point of care or near patient testing. Cou- antibodies, nucleic acids, and aptamers are expanding the
pling electrochemical or optical transducers together with role of biological recognition elements in biosensors. These
chemical or biological recognition elements has expanded molecules, when coupled to electrochemical, optical, and
the role of chemical sensors for measurement of analytes other transducers, produce biosensors for sensitive detection
without direct electrochemical or optical activity. of biomarkers for cancer, cardiac disease, and genetic testing.
Although there are several commercial applications of affinity
Content sensors in clinical use, continued growth in research publica-
This chapter reviews fundamental aspects of electrochemical tions and patents points to future possibilities for expanding
and optical measurements and their applications in routine applications to other clinical markers. This chapter discusses
clinical practice for measurements of blood gases (pH, partial nanotechnology to further enhance sensitivity of biosensors.
pressure of oxygen [PO2], partial pressure of carbon dioxide The sensor technologies routinely applied for in vitro measure-
[PCO2]), electrolytes (sodium [Na1], potassium [K1], cal- ments of critical care analytes (blood gases, glucose) are being
cium [Ca21], chloride [Cl2], magnesium [Mg21], lithium adapted for in vivo and minimally invasive or noninvasive
[Li1]), hematocrit [HCT], and toxic metals (lead [pb21]) di- wearable applications, through developing solutions to address
rectly in whole blood. The base principles of these electro- problems such as biocompatibility and calibration stability.
chemical and optical sensors are applied as building blocks Commercial products for continuous glucose monitoring
for enzyme-based biosensors for measurement of important (CGM) and health vital sign monitoring are a few examples of
metabolites directly in whole blood (glucose, lactate, urea, wearable sensors that are increasingly available.

*The full version of this chapter is available electronically on ExpertConsult.com.

a
The author gratefully acknowledges the contributions of Drs. Paul D’Orazio, Richard A. Durst, Ole Siggard-Andersen, and Mark E. Meyerhoff
for earlier versions of this chapter.

224
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CHAPTER 17 Electrochemistry and Chemical Sensors 224.e1

High input
INTRODUCTION impedance voltmeter
Recent advances in electromechanical, microfluidic, opti-
cal, and computer technologies have enabled the wider
adoption of chemical sensors utilizing electrochemical or
optical transduction methodologies (and associated bio-
sensors) in clinical analysis systems. Sensors for measure-
ment of blood gases, electrolytes, metabolites, trace metals,
Ag/AgCl Ag/AgCl
and other important biomarkers are incorporated into
automated, point-of-care, and laboratory clinical analyzers Inner
(see Chapters 29, 30, and 37, respectively). Miniaturization electrolyte
of sensors and the enabling technologies together with in-
Ion-selective KCI
creasing demand for analyzers that are easy to use, portable, frit
have wireless capability, and offer low-skilled operation membrane
with minimal process steps are driving the growth of point- SAMPLE
of-care testing.1 Biosensors that incorporate the same sens-
ing principles together with bioelements (such as enzymes, FIGURE 17.1 Schematic of ion-selective membrane electrode–
based potentiometric cell. Ag/AgCl, Silver-silver chloride; KCl,
antibodies, aptamers, nucleic acids, etc.) have also been
potassium chloride.
successfully applied for expanding the capabilities of these
devices to measure or monitor different metabolites, coagu-
lation reactions, biomarkers, detecting drugs, or toxic
chemicals through ultrasensitive immunoassays or genetic solutions is the unknown or test solution; this solution may
sequences in whole blood, plasma, serum, or urine sam- be replaced by an appropriate reference solution for calibra-
ples. When integrated into chromatographic systems (see tion purposes. By convention, the cell notation is shown so
Chapter 19), electrochemical detectors provide a highly that the left electrode (ML) is the reference electrode, and the
sensitive and selective means of detecting a variety of other right electrode (MR) is the indicator (measuring) electrode
analytes, such as therapeutic drugs, neurotransmitters, glu- (see Eq. 17.3).2
tathione, and homocysteine. In addition, the development The electromotive force (E or EMF) is defined as the
and application of optodes, which are based on some of the maximum difference in potential between the two electrodes
same selective chemistries used in electrochemical devices, (right minus left) obtained when the cell current is zero. The
have resulted in another analytical tool for measuring cell potential is measured using a potentiometer, of which the
blood gases and electrolytes. common pH meter is a special type. The direct-reading po-
In this chapter, the fundamental electrochemical principles tentiometer is a voltmeter that measures the potential across
of (1) potentiometry, (2) voltammetry and/or amperometry, the cell (between the two electrodes); however, to obtain an
(3) conductance, and (4) coulometry are summarized, and accurate potential measurement, it is necessary that no cur-
their clinical applications are presented. Optodes and biosen- rent flow through the cell. This is accomplished by incorpo-
sors are also discussed. The chapter concludes with a discus- rating high resistance within the voltmeter (input impedance
sion of in vivo, minimally invasive, and noninvasive sensors greater than 1012 V). Modern direct-reading potentiometers
that are growing in clinical use for continuous glucose moni- are accurate and can be modified to provide direct digital
toring (CGM) or other wearable devices. displays or printouts.
Within any one conductive phase, the potential is
POTENTIOMETRY AND ION-SELECTIVE constant as long as the current flow is zero. However, a
potential difference arises between two different phases in
ELECTRODES contact with each other. The overall potential of an electro-
Potentiometry is widely used clinically for the measurement chemical cell is the sum of all potential gradients that exist
of pH, PCO2, and electrolytes (Na1, K1, Cl2, Ca21, Mg21, between different phases of the cell. The potential of a
Li1) in whole blood, serum, plasma, and urine, and as the single electrode with respect to the surrounding electrolyte
basis for some biosensors for metabolites of clinical interest and the absolute magnitude of the individual potential
(urea or blood urea nitrogen [BUN]). gradients between phases are unknown and cannot be
measured. Only potential differences between two elec-
Basic Concepts trodes (half-cells) can be measured. Potential gradients can
Potentiometry is the measurement of an electrical potential be classified as (1) redox potentials, (2) membrane poten-
difference between two electrodes (half-cells) in an electro- tials, or (3) diffusion potentials. Generally, it is possible to
chemical cell (Fig. 17.1) when the cell current is zero (gal- devise a cell in such a manner that all potential gradients
vanic cell). Such a cell consists of two electrodes (electron except one are constant. This potential then can be related
and metallic conductors) that are connected by an electro- to the activity of a specific ion of interest (e.g., hydrogen
lyte solution (ion conductor). An electrode, or half-cell, [H1] or Na1).
consists of a single metallic conductor that is in contact with
an electrolyte solution. Ion conductors are composed of one Types of Electrodes
or more phases that are either in direct contact with each Many different types of electrodes are used for potentiomet-
other or separated by membranes permeable only to specific ric applications. They include redox, ion-selective membrane
cations or anions (see Fig. 17.1). One of the electrolyte (glass and polymer), and PCO2 electrodes.

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224.e2 SECTION II Analytical Techniques

Redox Electrodes (platinized) with highly porous Pt (Pt black) to catalyze the
Redox potentials are the result of chemical equilibria involv- electrode reaction:
ing electron transfer reactions: 1
H e ↔ H2 (17.5)
Oxidized form (Ox) 1 ne2 6 Reduced form (Red) (17.1) 2
where n 5 the number of electrons involved in the reaction. The electrode potential is given by
Any substance that accepts electrons is an oxidant (Ox), and (fH )
12
any substance that donates electrons is a reductant (Red). 0
E E N log 2 (17.6)

The two forms, Ox and Red, represent a redox couple (conju- aH
gate redox pair). Usually, homogeneous redox processes take or
place only between two redox couples. In such cases, elec-
E E 0 N  log ( f H2 ) log aH  (17.7)
12
trons are transferred from Red1 to an Ox2. In this process,
Red1 is oxidized to its conjugate Ox1, whereas Ox2 is reduced  
to Red2: where E0 5 0 at all temperatures (by convention); fH2 5 fugacity
of H2 gas; aH1 5 activity of H1 ions; and 2log aH1 5 negative
Red1 1 Ox2 6 Ox1 1 Red2 (17.2)
log of H1 activity (paH1 or pH).
In an electrochemical cell, electrons may be accepted from When the partial pressure of H2 (pH2) in the solution (and
or donated to an inert metallic conductor (e.g., platinum hence, fH2) is maintained constant by bubbling H2 through the
[Pt]). A reduction process tends to charge the electrode posi- solution, the potential is a linear function of log aH1 (5 2pH).
tively (remove electrons), and an oxidation process tends to In the standard H2 electrode, the electrolyte consists of
charge the electrode negatively (add electrons). By convention, an aqueous solution of HCl with aHCl equal to 1.000 (or
a heterogeneous redox equilibrium (Eq. 17.2) is represented cHCl 5 1.2 mol/L) in equilibrium with a gas phase, and with
by the cell: fH2 equal to 1.000 (or pH2 5 101.3 kPa 5 1 atm). The standard
H2 electrode is also used as a reference electrode.
ML | Red1 2 Ox2 YY Ox2 2 Red2 | MR (17.3)
Metal electrodes participating in Redox reactions. The silver-
A positive potential (E . 0) for this cell signifies that the silver chloride (Ag/AgCl) electrode is an example of a metal
cell reaction proceeds spontaneously from left to right; E , 0 electrode that participates as a member of a redox couple. The
signifies that the reaction proceeds from right to left, and Ag/AgCl electrode consists of an Ag wire or rod coated with
E 5 0 indicates that the two redox couples are at mutual AgCl(solid) in contact with a Cl2 solution of constant activity;
equilibrium. this sets the half-cell potential. The Ag/AgCl electrode is itself
The electrode potential (reduction potential) for a redox considered a potentiometric electrode because its phase bound-
couple is defined as the couple’s potential measured with re- ary potential is governed by an oxidation-reduction electron
spect to the standard H2 electrode, which is set equal to zero transfer equilibrium reaction that occurs at the surface of Ag:
(see later discussion of the H2 electrode). This potential, by
AgCl(solid) 1 e2 6 Ag°(solid) 1 Cl2 (17.8)
convention, is the EMF of a cell, where the standard H2 elec-
trode is the reference electrode (left electrode) and the given The Nernst equation for the reference half-cell potential of
half-cell is the indicator electrode (right electrode). The re- an Ag/AgCl reference electrode is written as follows:
duction potential for a given redox couple is shown by the RT aAgCl
Nernst equation: 0
E Ag AgCl E Ag ln (17.9)
AgCl
nF aAg aCl
E E 0 N log aRed E 0 0.0592 V log aRed (17.4) Because AgCl and Ag are both solids, their activities are
n aOx n aOx equal to unity (aAgCl 5 a0Ag 5 1). Therefore from Eq. (17.9),
the half-cell potential is controlled by the activity of the Cl2
where E 5 electrode potential of the half-cell; E0 5 standard ion in solution (aCl2) contacting the electrode.
electrode potential when aRed/aOx 5 1; n 5 number of electrons The Ag/AgCl electrode is used both as an internal reference
involved in the reduction reaction; N 5 (R 3 T 3 ln 10)/F element in potentiometric ion-selective electrodes (ISEs) and
(the Nernst factor if n 5 1); N 5 0.0592 V if T 5 298.15 K as an external reference electrode half-cell of constant poten-
(25 °C); N 5 0.0615 V if T 5 310.15 K (37 °C); R 5 gas constant tial, which is required to complete a potentiometric cell (see
(5 8.31431 J 3 K21 3 mol21); T 5 absolute temperature (in Fig. 17.1). In both cases, the Ag/AgCl electrode must be in
kelvins); F 5 Faraday constant (5 96,487 Coulombs 3 mol21); equilibrium with a solution of constant Cl2 ion activity.
ln 10 5 natural logarithm of 10 5 2.303; a 5 activity; The Ag/AgCl element of the external reference electrode
and aRed/aOx 5 product of mass action for the reduction half-cell is in contact with a high-concentration solution of a
reaction. soluble Cl2 salt. Saturated KCl is commonly used. A porous
Redox electrodes currently in use include (1) inert metal membrane or frit is frequently employed to separate the con-
electrodes immersed in solutions containing redox couples centrated KCl from the sample solution. The frit serves both
and (2) metal electrodes whose metal functions as a member as a mechanical barrier to hold the concentrated electrolyte
of the redox couple. within the electrode and as a diffusional barrier to prevent
Inert metal electrodes. Platinum and gold (Au) are exam- proteins and other species in the sample from coming into
ples of inert metals used to record the redox potential of a contact with the internal Ag/AgCl element, which could poi-
redox couple dissolved in an electrolyte solution. The H2 son and alter its potential. The interface between two dis-
electrode is a special redox electrode for pH measurement. It similar electrolytes (concentrated KCl/calibrator or sample)
consists of a Pt or Au electrode that is electrolytically coated occurs within the frit and develops the liquid–liquid junction

potential (Ej), a source of error in potentiometric measure- The Nikolsky-Eisenman equation describes the selectivity
ments. The difference in Ej between the calibrator and sample of an ISE for the ion of interest over interfering ions:
 2.303 RT 
( )
(residual liquid junction potential) is responsible for this er-
 log ai ∑ K i j a j
zi z j
ror and can be minimized and usually neglected in practice if E E 0  (17.11)
 z F 
the compositions of the calibrating solutions are matched as i

closely as possible to the sample with respect to ionic content where ai 5 activity of the ion of interest; aj 5 activity of
and ionic strength. An equitransferrant electrolyte at high the interfering ion; and Ki/j 5 selectivity coefficient for the
concentration as the reference electrolyte further helps to primary ion over the interfering ion. Low values indicate
minimize the residual liquid junction potential. KCl at a con- good selectivity for the analyte i over the interfering ion j;
centration 2 mol/L or more is preferred. Differences of ap- zi 5 charge of the primary ion; and zj 5 charge of the inter-
proximately 22% in the measurement of sodium by ISEs fering ion.
have been demonstrated when the KCl concentration in the All other terms are identical to those in the Nernst equa-
reference electrolyte is lowered from 3 to 0.5 mol/L.3 The tion (Eq. 17.4).
magnitude of the residual liquid junction potential may also Various approaches may be used to determine the selectiv-
be estimated by the Henderson equation4 with sufficient ity of an ISE for a primary ion over an interfering ion.6,7
knowledge of ionic activities, ionic charges, and ionic mo- A straightforward approach is the separate solution method,
bilities for each electrolyte on both sides of the junction and in which the potential of an ISE is determined in solutions
the temperature. Using this estimate, a correction to the over- of the primary and interfering ions separately, but at equal
all cell potential may be applied. ionic activities. The selectivity coefficient is then calculated
The presence of erythrocytes in the sample may affect the as follows:
magnitude of the residual liquid junction potential in a less E j Ei  z 
predictable manner. For example, erythrocytes in blood of log K ij  1 i  log ai (17.12)
2.303 RT  zj 
normal hematocrit are estimated to produce approximately
1.8 mmol/L positive error in the measurement of Na by ISEs nF
when an open, unrestricted liquid–liquid junction is used.5 An alternate approach to determine selectivity coefficient
This bias may be minimized if a restrictive membrane or frit is the fixed interference method, in which the potential re-
is used to modify the liquid–liquid junction. sponse of an ISE to the primary ion is determined in solu-
The saturated calomel electrode is another example of a tions of constant activity of the interfering ion. The potential
metal electrode that participates as a member of a redox values obtained are plotted versus the logarithm of the activ-
couple. The calomel electrode consists of mercury (Hg) cov- ity of the primary ion ai. The intersection of the extrapola-
ered by a layer of relatively insoluble calomel (Hg2Cl2) (or tion of the linear portions of this plot indicates the value of
present as insoluble salt dispersed in the electrolyte), which is ai to be used to calculate Ki/j :
in contact with an electrolyte solution containing Cl2. The z zj
K i j 5 ai a j i (17.13)
oxidation-reduction equilibrium reaction is as shown:

Hg2Cl2 1 2e 6 2Hg° 1 2Cl
2 2
(17.10)
where all terms have the same definition as in Eq. (17.12).
As with the Ag/AgCl electrode, the half-cell potential is Traditionally, the fixed interference method has been pre-
controlled by the activity of Cl2 ion contacting the electrode. ferred because it more closely resembles a practical applica-
Calomel electrodes are frequently used as reference electrodes tion of the sensor, in that primary and interfering ions are
for pH measurements using glass pH electrodes but are not present simultaneously in solution and must compete for
commonly used in clinical instrumentation using electro- complexation sites in the ISE membrane.
chemical sensors. Most ISEs used in clinical practice have sufficient selectiv-
ity and do not require correction for interfering ions. Oesch
Ion-Selective Electrodes and colleagues8 have published required ISE selectivity coef-
Membrane potentials are caused by the permeability of cer- ficients for ions commonly measured in clinical chemistry
tain types of membranes to selected anions or cations. Such over other ions found in blood. Table 17.1 shows required
membranes are used to fabricate ISEs that selectively interact selectivity coefficients for the measurement of cations of in-
with a single ionic species. The potential produced at the terest in clinical chemistry over potentially interfering cat-
membrane–sample solution interface is proportional to the ions, assuming an acceptable maximum interference of 1%
logarithm of the ionic activity or concentration of the ion in for the ion of interest.
question. Measurements with ISEs are simple, often rapid, Glass membrane and polymer membrane electrodes are
nondestructive, and applicable to a wide range of concentrations. two types of ISEs that are commonly used in clinical chemis-
The ion-selective membrane is the “heart” of an ISE be- try applications.
cause it controls the selectivity of the electrode. Ion-selective The glass electrode. Glass membrane electrodes are used
membranes are typically composed of glass, crystalline, or to measure pH and Na1, and as an internal transducer for
polymeric materials. The chemical composition of the mem- PCO2 sensors. The H1 response of thin glass membranes was
brane is designed to achieve an optimal permselectivity to- first demonstrated in 1906 by Cremer.9 In the 1930s, practical
ward the ion of interest. In practice, other ions exhibit finite application of this phenomenon for measurement of acidity
interaction with membrane sites and display some degree of in lemon juice was made possible by the invention of the pH
interference for determination of an analyte ion. In clinical meter by Beckman.2 Glass electrode membranes are formu-
practice, if the interference exceeds an acceptable quantity, a lated from melts of silicon and/or aluminum oxide (Al2O3)
correction is required. mixed with oxides of alkaline earth or alkali metal cations. By

200
TABLE 17.1 Required Selectivities for
Cation-Selective Ion-Selective Electrodes
150
for Whole Blood, Plasma, and Serum

Millivolts vs. Ag/AgCl

Measurements 100
REQUIRED SELECTIVITY COEFFICIENT
Primary (LOGKI/J) FOR INTERFERING CATION (J) 50
Ion (i) H 1
Li 1
Na 1
K 1
Mg 21
Ca 21
0
H1 — 26.5 28.5 27.0 27.7 27.7
Li1a 2.1 — 24.3 22.8 23.5 23.6 50
Na1 4.4 20.1 — 20.6 21.2 21.3
K1 2.8 21.7 23.6 — 22.8 22.9 100
Mg21 8.9 0.1 23.9 20.9 — 22.4 6 5 4 3 2 1
Ca21 9.3 0.4 23.6 20.6 21.9 — Log K activity, mol/L
a
Assumes a therapeutic range for Li1 between 0.7 and 1.5 mmol/L. FIGURE 17.2 Typical electromotive force (EMF) response of
Ca, Calcium; H, hydrogen; K, potassium; Li, lithium; Mg, magne- potassium (K1) selective membrane electrode to changes in
sium; Na, sodium. activity of K1 in the sample solution. Bracketed interval repre-
sents the normal reference interval of K1 concentration in
blood. Ag/AgCl, Silver-silver chloride. (From D’Orazio P. In:
varying the glass composition, electrodes with selectivity for Lewendrowski K, editor. Clinical chemistry: laboratory manage-
ment and clinical correlations. Philadelphia: Lippincott, Williams
H1, Na1, K1, Li1, rubidium (Rb1), cesium (Cs1), Ag1, thallium
and Wilkins; 2002:455.)
(Tl1), and ammonium (NH41) have been demonstrated.10
However, glass electrodes for H1 and Na1 are currently the
only types with sufficient selectivity over interfering ions to
allow practical application in clinical chemistry. A typical colleagues that the neutral antibiotic valinomycin could be
formulation for H1 selective glass is 72% silicon dioxide (SiO2), incorporated into organic liquid membranes (and later plasti-
22% Na2O, and 6% calcium oxide (CaO), which has a selec- cized PVC membranes), resulting in a sensor with high selec-
tivity order of H1 . Na1 . K1. This glass membrane has tivity for K1 over Na1 (KK/Na 5 2.5 3 1024).16 The K1 ISE
sufficient selectivity for H1 over Na1 to allow error-free mea- based on valinomycin was the first example of a neutral carrier
surements of pH in the range of 7.0 to 8.0 ([H1] 5 1027 to ISE and is used extensively today for the routine measurement
1028 mol/L) in the presence of . 0.1 mol/L Na1. Glass pH of K1 in blood. Fig. 17.2 shows the response of the valinomy-
electrodes with selectivity coefficients (KH/Na) over Na1 of cin-based K1 ISE in the presence of physiologic concentrations
1027 and better have been realized. By altering the formula- of Na1, Ca21, and Mg21. The wide linear range and excellent
tion of the glass membrane slightly to 71% SiO2, 11% Na2O, selectivity of this ISE over three orders of magnitude makes it
and 18% Al2O3, its selectivity order becomes H1 . Na1 . K1. suitable for the measurement of K1 in blood and urine. The
Thus the preference of the glass membrane for H1 over Na1 K1 range in blood is only a small portion of the electrode lin-
is greatly reduced, resulting in a practical sensor for Na1 at ear range and is spanned by a total DEMF of approximately 9
pH values typically found in blood.11 mV. Interference from other cations, which is seen as deviation
Polymer membrane electrodes. Polymer membrane ISEs from linearity, is not apparent at K1 activities more than 1024
are used for monitoring pH and for measuring electrolytes, mol/L. Other, less selective polymer-based ISEs (e.g., for the
including K1, Na1, Cl2, Ca21, Li1, Mg21, and carbonate measurement of Mg21 and Li1) are subject to interference
(CO322) (for total CO2 measurements). They are the pre- from Ca21 and Na1 and Na1, respectively, requiring simulta-
dominant class of potentiometric electrodes used in modern neous determination and correction for the presence of sig-
clinical analysis instruments. nificant concentrations of these interfering ions.17,18
Mechanisms of response of these ISEs fall into three cate- Studies regarding the relationship between molecular struc-
gories: (1) charged, dissociated ion exchanger; (2) charged ture and ionic selectivity have resulted in the development of
associated carrier; and (3) neutral ion carrier (ionophore).12,13 polymer-based ISEs using a number of naturally occurring
An early charged-associated, ion-exchanger type ISE for Ca21 and synthetic ionophores, with sufficient selectivity for appli-
was developed and commercialized for clinical application cation in clinical analysis. The chemical structures of several of
in the 1960s based on the Ca21-selective, ion-exchange/ these neutral ionophores are illustrated in Fig. 17.3.
complexation properties of 2-ethylhexyl phosphoric acid dis- Dissociated anion exchanger-based electrodes using lipo-
solved in dioctyl phenyl phosphonate.14 A porous membrane philic quaternary ammonium salts as active membrane com-
was impregnated with this cocktail and mounted at the end ponents are still used commercially for the determination of
of an electrode body. This type of sensor was referred to as Cl2 in whole blood, serum, and plasma, despite some limita-
the “liquid membrane” ISE. Later, a method was devised in tions.19 Selectivity for this type of ISE is controlled by extrac-
which these ingredients could be cast into a plasticized tion of the ion into the organic membrane phase and is a
poly(vinyl chloride) (PVC) membrane that was more rugged function of the lipophilic character of the ion (because,
and convenient to use. This same approach is still used today unlike the carriers described earlier, no direct binding inter-
to formulate PVC-based ISEs for clinical use.15 action occurs between the exchanger site and the anion in
A major breakthrough in the development and routine ap- the membrane phase). Thus the selectivity order for a Cl2
plication of PVC-type ISEs was the discovery by Simon and ISE based on an anion exchanger is fixed as lipophilic anion

H
N N
H O
N
O
O O O
O
Tridodecylamine: H
O H
O N
O O R1 O
N O
H O O O O
O CH3
O O O
O O O
O H3C O
N O O O
O H
N
O R3
H
Nonactin: NH4
Valinomycin: K

H3C H3C
H3C
HO CH3 H
N CH3
H 3C O O O
O H H H O
O H3C—O
H OH OH
O
H3C CH3 Methyl monensin: Na
N
O OCH3
O O
O
O O
O

N O O

ETH 227: Na

O O
N
O O O O
O
O O O O
O
Bis(benzyl-15-crown-5)-heptanedoate: K
N

ETH 1117: Mg2

O
N
O
O
O
O O
N N O
O O
O
O
N
O
ETH 157: Na ETH 1001: Ca2
FIGURE 17.3 Structures of common ionophores used to fabricate polymer membrane–type ion-selective
electrodes for clinical analysis.

R2 . perchlorate (ClO42) . iodide (I2) . nitrate (NO32) . the analyte ion for extended periods of time, and the con-
bromide (Br2) . Cl2 . fluoride (F2), where R2 represents centration of the analyte ion in the internal solution should
anions with greater lipophilic character than ClO42. Applica- be low.26 To avoid leakage of primary ions from the inner
tion of the Cl2 ion-exchange electrode is therefore limited to solution of conventional polymer membrane ISEs, new,
samples without significant concentrations of anions more more stable designs for solid-state ion sensors have been
lipophilic than Cl2. For example, blood samples containing suggested, in which the ionophore-doped, polymer ion–
salicylate or thiocyanate will produce positive interference for sensing membrane (based on a more water-repellent
the measurement of Cl2. Repeated exposure of the electrode poly[methylmethacrylate]/poly[decylmethacrylate copoly-
to the anticoagulant heparin will lead to loss of electrode mer]) is coated onto a conductive poly(3-octylethiophene
sensitivity toward Cl2 because of the extraction of negatively 2,5-diyl) (POT) polymer layer on the surface of an under-
charged heparin into the membrane. This extraction process lying Au electrode.27
has been used successfully to devise a method to detect heparin An interesting application of Na1 selective polymer (or
concentrations in blood by potentiometry20 and to develop a glass) membrane electrodes is seen in the determination of
simple potentiometric technique to screen for the presence of whole blood hematrocrit.28 Because intracellular Na1 con-
toxic, high-charge density polyanion contaminants (e.g., over- centrations are much lower than those in the plasma phase,
sulfated chondroitin sulfate) in biomedical-grade heparin the change in Na1 concentration (dilution) measured poten-
preparations.21 tiometrically before and after erythrocyte lysis can be used to
High selectivity for the CO322 anion can be achieved using assess the hematocrit of the blood sample. This approach can
a neutral carrier ionophore that possesses trifluoroacetophe- be coupled with simultaneous measurement of changes in K1
none groups doped within a polymeric membrane.22,23 Such ion concentration as determined with a valinomycin-based
ionophores form negatively charged adducts with CO322 an- polymer membrane ISE to quantify the concentration of K1
ions, and the resulting electrodes have proven useful in com- ions within red blood cells.29
mercial instruments for determination of total CO2 in serum Recently, a reversible polymer membrane ISE for mea-
and/or plasma, after dilution of the blood to a pH value in the surement of polyions was described.30 The sensor consists of
range of 8.5 to 9.0, where a significant fraction of total CO2 a highly lipophilic electrolyte (tetradodecylammonium-
will exist as CO322 anions. dinonylnaphthalenesulfonate), which is free of intrinsic ion-
A typical formulation of a PVC membrane ISE used in exchange properties, added to a plasticized PVC membrane
clinical instrumentation consists of the following in % by at high concentration (approximately 10 wt%). Application
weight (wt%): of a cathodic current pulse across the membrane results in
1 to 3 wt% ionophore; reversible extraction and potentiometric response to the
≈ 64 wt% plasticizer; polycation protamine. Protamine is a polypeptide adminis-
≈ 30 wt% PVC; and ,1 wt% additives. tered to neutralize heparin activity. A response to heparin in
The plasticizer is crucial in controlling the polarity of the whole blood was demonstrated via protamine titration. Later,
membrane, and thus along with the ionophore, plays a piv- it was found that by changing the lipophilic cation in the
otal role in determining the selectivity of the membrane membrane to tridodecylmethylammonium, application of an
toward the ion of interest. A large lipophilic anion (e.g., anodic current pulse resulted in a direct potentiometric re-
tetraphenylborate derivative) is often included as an addi- sponse to heparin.31 Testing of other polyanions in addition
tive for preparation of cation-selective ISE membranes. to heparin led to the conclusion that the magnitude of the
This anion serves as a counter-anion for the cation of inter- potentiometric response is a function of charge density of the
est as it is extracted into the membrane phase, forming a polyanion.
positively charged complex with the neutral ionophore. Electrodes for partial pressure of carbon dioxide. Elec-
However, it is the ratio of the bound-to-unbound iono- trodes have been developed to measure PCO2 in body fluids.
phore sites at the membrane surface that determines the The first PCO2 electrode, developed in the 1950s by Stow and
magnitude of the phase boundary potential generated by Severinghaus, used a glass pH electrode as the internal ele-
the ISE membrane.24 Thus the selective response to the ac- ment in a potentiometric cell for measurement of the PCO2.32
tivity of the ion of interest is an interfacial property of the This important development paved the way for commercial
given ISE membrane. availability of the three-channel blood analyzer (pH, PCO2,
Studies have demonstrated that the ultimate detection PO2) which gives the complete picture of the oxygenation
limits of polymer membrane–type ISEs are controlled in and acid-base status of blood.
part by the leakage of analyte ions from the internal solu- Fig. 17.4 shows a diagram of a typical Severinghaus-
tion to the outer surface of the membrane and into the style electrode for PCO2. A thin membrane (approximately
sample phase in close contact with the membrane.25 Hence, 20 mm), permeable only to gases and water vapor, is in
lower limits of detection can be achieved by decreasing the contact with the sample. Membranes of silicone rubber,
concentration of the primary analyte ion within the inter- Teflon, and other polymeric materials are suitable for this
nal solution of the electrode. Furthermore, this leakage of purpose. On the opposite side of the membrane is a thin
analyte ions, coupled with an ion exchange process at the electrolyte layer consisting of a weak bicarbonate salt
membrane sample interface when the selectivity of the (approximately 5 mmol/L) and a Cl2 salt. A pH electrode
membrane over other ions is assessed, can often yield a and an Ag/AgCl reference electrode are in contact with
measured potentiometric selectivity coefficient that under- this solution. The PCO2 electrode is a self-contained po-
estimates the true selectivity of the membrane. To deter- tentiometric cell. CO2 gas from the sample or calibration
mine “unbiased” selectivity coefficients by the separate matrix diffuses through the membrane and dissolves in
solution method, the membrane should not be exposed to the internal electrolyte layer. Carbonic acid is formed and

Glass electrode shaft

Plastic holster

Electrode housing

Reference electrode (Ag/AgCl) Internal electrode (Ag/AgCl)

Sodium bicarbonate Phosphate buffer

O-ring

Sample inlet Sample outlet

pH-sensitive glass membrane

Porous spacer
CO2-permeable membrane (silicone rubber)
Cuvet
Glass window
FIGURE 17.4 Schematic of severinghaus-style partial pressure of carbon dioxide (PCO2) sensor
used to monitor CO2 concentrations in blood samples. Ag/AgCl, Silver-silver chloride. (From Siggard-
Andersen O. The acid-base status of the blood. 4th ed. Baltimore, MD: Williams & Wilkins; 1974:172.)

dissociates, shifting the pH of the bicarbonate solution in pH CO2

PVC
the internal layer as follows: membrane
(H)o
CO2 1 H2O 8 H2CO3 6 H1 1 HCO23 (17.14) CO2 CO2

and

D log PCO2(sample) ≈ D pH(internal layer) (17.15) Strong buffer
(H)i (H)i
The relationship between the sample PCO2 and the signal Bicarbonate-containing
generated by the internal pH electrode is logarithmic and is solution
governed by the Nernst equation. The electrode may be cali- EpH Internal
electrode ECO2 EpH
brated using precision gas mixtures or using solutions with
(Ag/AgCl)
stable PCO2 concentrations. Although Severinghaus-style
electrodes for PCO2 have gained widespread use in modern
blood gas analyzers, the format in which such sensors may be FIGURE 17.5 Differential planar partial pressure of carbon diox-
constructed is limited by size, shape, and the ability to fabri- ide (PCO2) potentiometric sensor design, based on two identical
cate the internal pH sensitive element. polymeric membrane pH electrodes, but with different internal
A slightly different potentiometric cell for PCO2 is shown reference electrolyte solutions. Both pH sensing membranes
are prepared with a hydrogen (H1)-selective ionophore. Ag/AgCl,
in Fig. 17.5. This cell arrangement uses two PVC-type, pH-
Silver-silver chloride; PVC, poly(vinyl chloride).
selective electrodes in a differential mode. The electrode
membranes contain a lipophilic amine-type neutral iono-
phore that exhibits high selectivity for H1 (see Fig. 17.3). One The signal at the electrode with the buffered internal layer is
electrode has an internal layer that is buffered, and the other unaffected by the CO2 that diffuses across the membrane.
is unbuffered, consisting of a low concentration of bicarbon- Consequently, one half of the sensor responds to pH alone,
ate salt. CO2 gas from the sample or calibration matrix dif- and the other half responds to both pH and PCO2. The signal
fuses across the outer H1-selective PVC membranes of both difference between the two electrodes cancels any contribu-
sensors. On the unbuffered side, CO2 diffusion produces a tion of sample pH to the overall measured cell potential. The
potential shift at the internal interface of the pH-responsive differential signal is proportional only to PCO2. Unlike the
membrane proportional to the sample PCO2 concentration. traditional Severinghaus-style electrode, this differential

potentiometric cell PCO2 sensor has been commercialized in increase in ionic strength. This effect is more pronounced
a planar format and is more easily adaptable to mass produc- when the charge (z) of the ion is higher. Activity coefficients
tion in sensor arrays.33 for ions in biological fluids, such as blood and serum, are dif-
ficult to calculate with accuracy because of the uncertain
Direct Potentiometry by Ion-Selective Electrodes: Units contribution of macromolecular ions, such as proteins, to the
of Measure and Reporting for Clinical Applications overall ionic strength. However, assuming that the normal
Older analytical methods such as flame photometry for the ionic strength of blood plasma is 0.160 mol/kg, estimates of
measurement of electrolytes provide the total concentration activity coefficients at 37 °C are as follows: Na1 5 0.75, K1 5
(c) of a given ion in the sample, usually expressed in units of 0.74, and Ca21 5 0.31. Referring to Eq. (17.17), activity and
millimoles of ion per liter of sample. Molality (m) is a mea- concentration will differ greatly in samples of physiologic
sure of the moles of ion per mass of water (millimoles per ionic strength, especially for divalent ions.
kilogram) in the sample. Using the Na1 ion as an example, the Physiologically, ionic activity is assumed to be more rele-
relationship between concentration and molality is given by vant than concentration when chemical equilibria or biologi-
cal processes are considered. Practically, however, ionic con-
cNa1 5 mNa1 3 rH2O (17.16)
centration is the more familiar term in clinical practice,
where rH2O 5 mass concentration of water in kilograms per forming the basis of reference intervals and medical decision
liter. For normal blood plasma, the mass concentration of concentrations for electrolytes. Early in the evolution of ISEs
water is approximately 0.93 kg/L, but in specimens with in- as practical tools in clinical chemistry, it was decided that
creased lipids or protein, the value may be as low as 0.8 kg/L. changing clinical reference intervals to a system based on
In these specimens, the difference between concentration and activity instead of concentration was impractical and carried
molality may be as great as 20%. A significant advantage of the risk for clinical misinterpretation. A pragmatic approach
direct potentiometry by ISE for the measurement of electro- for using ISEs in modern analyzers without changing estab-
lytes is that the technique is sensitive to molality, and there- lished concentration-based reference intervals is to formulate
fore is not affected by variations in the concentration of calibration solutions with ionic strengths and ionic composi-
protein or lipids in the sample. Techniques such as flame tions as close as possible to those of blood plasma. In this way,
photometry, ISE methods used in laboratory clinical chemis- the activity coefficient of each ion in the calibrating solutions
try analyzers that require sample dilution (also called indirect approximates that in the sample matrix, which allows cali-
potentiometry), and other photometric methods that require bration and measurement of electrolytes in units of concen-
sample dilution are affected by the presence of protein and tration instead of activity.35
lipids. In these methods, only the water phase of the sample A typical set of solutions for multi-ISE calibration in an
is diluted, which produces results lower than molality as a analyzer is shown in Table 17.2. Two points are used to cali-
function of the concentration of protein and lipids in the brate each ISE. The difference in the cell potential generated
sample. Thus there is a risk for errors, such as a falsely low by these two solutions (DE) is used to calculate the response
Na1 concentration (pseudohyponatremia), in cases of ex- slope of the cell (slope 5 DE/Dlog c), where c is the concen-
tremely increased protein and lipid concentrations (see also tration of ion in each calibrating solution, substituted for
Chapter 37).34 Such falsely low Na1 results can be problem- activity. The standard electrode potential, E0, is calculated as
atic for users where both direct ISE (point of care, whole the y-intercept. Determination of the ion concentration in an
blood based) and indirect ISE (laboratory, plasma/serum unknown sample is then a straightforward solution of the
based) measurements are routinely used interchangeably Nikolsky-Eisenman equation (Eq. 17.11), after the cell poten-
within a facility. tial generated by the sample is measured. The measured slope
In addition to the difference between molality and con- is used in place of the 2.303RT/ziF term of Eq. (17.11). In the
centration, measurement of ions by direct potentiometry absence of significant influence from interfering ions on
provides yet another unit of measurement known as activity measurement of the primary ion (e.g., # 1% interference on
(a), the concentration of free, unbound ion in solution. Un- the measured value), contributions from aj in Eq. (17.11)
like methods sensitive to ion concentration, ISEs do not sense may be ignored.
the presence of complexed or electrostatically “hindered”
ions in the sample. The relationship between activity and
concentration, using Na1 ion as an example, is expressed as TABLE 17.2 Examples of Two-Value
Calibrating Solutions for Measurement of
aNa1 5 gNa1 3 cNa1 (17.17) pH and Electrolytes by Direct Potentiometry
where g 5 dimensionless quantity known as the activity co- Expected
efficient. The activity coefficient is primarily dependent on Calibration Slope Point Signal D,
ionic strength of the sample as described by the Debye- Analyte Point (mmol/L) (mmol/L) (mV)
Huckel equation:

log
(
A Z 2 I 05. ) (17.18)
Na1
K1
140
4.0
110
8.0
6.6
18
1 B a I 05. Ca21 1.25 2.50 9
where A and B 5 temperature-dependent constants (A 5 Cl2 100 80 6
0.5213 and B 5 3.305 in water at 37 °C); a 5 ion size param- pH 7.38 (pH units) 6.84 (pH units) 32.4
eter for a specific ion; and I 5 ionic strength (I 5 0.5 Sm 3 Ionic strength adjusted to 160 mmol/kg with buffer salts and
z2, where z is the charge number of the ions). Eq. (17.18) inert electrolytes. Ca21, Calcium; Cl2, chloride; K1, potassium;
shows that a decrease in the activity coefficient occurs with an Na1, sodium.

Calibration of the cell is done in units of concentration; E0 value for that reaction [standard reduction potential]) and
however, as mentioned earlier, direct potentiometry is sensi- the kinetics for heterogeneous electron transfer at the inter-
tive to the molality of the ion, which is related to concentra- face of the working electrode. Often, slow kinetics of electron
tion by the water content of the sample (Eq. 17.16). The wa- transfer for the redox reaction on a given inert working elec-
ter content of the aqueous calibrating solutions shown in trode (Pt, carbon, Au, and so on) mandates use of a much
Table 17.2 is approximately 0.99 kg/L. The water content of more negative (for reductions) or positive (for oxidations)
normal blood plasma is approximately 0.93 kg/L. Molality is Eappl than that predicted based merely on the E0 for a given
7% greater than the concentration in this normal plasma redox reaction. This is called an overpotential (h). Regardless
specimen. The direct potentiometric cell will report results of whether an overpotential for electron transfer exists, a
approximately 6% greater than the concentration in normal specific oxidation or reduction reaction occurs at the surface
specimens because of this difference in water content be- of the working electrode in voltammetry and/or amperome-
tween the sample and the calibrator (0.99/0.93 5 1.06). try, and it is the charge transfer at this interface (current flow)
Direct potentiometry presents an advantage in that the tech- that provides the analytical information.
nique is not affected by the presence of protein and lipids For electrolytic cells that form the basis of voltammetric
in the sample; however, the application of clinical reference and amperometric methods:
intervals based on concentration again poses a risk for confu-
Eappl 5 Ecell 1 h 2 iRcell (17.19)
sion and clinical misinterpretation. Most manufacturers of
electrolyte measurement systems have overcome this prob- where Ecell 5 thermodynamic potential between the working
lem in a practical way by following Clinical and Laboratory and reference electrodes in the absence of an applied external
Standards Institute (CLSI) guidelines that recommend the voltage. When the external voltage is greater or less than this
use of correlation factors to standardize ISE measurements to equilibrium potential, plus or minus any overpotential (h),
units of concentration. These factors may be obtained by then current will flow because of an oxidation or reduction
standardizing the ISE measurement to certified reference reaction at the working electrode. A voltammogram is simply
materials based on human serum, with electrolyte values as- the plot of observed current, i, versus Eappl (Fig. 17.6). In
signed in units of concentration.36–38 Appropriate correlation amperometry (see later), a fixed voltage is applied, and the
factors are then applied to sample calculations using algo- resulting current is monitored. The amount of current is in-
rithms resident in the instrument software. versely related to the resistance of the electrolyte solution and
to any “apparent” resistance that develops because of mass
POINTS TO REMEMBER transfer of the analyte species to the surface of the working
electrode. Because electrochemical reactions are heteroge-
Advantages of Electrochemical Sensors for Whole neous, occurring only at the surface of the working electrode,
Blood Measurements the amount of current observed is also highly dependent on
• Electrochemical sensors measure the most important the surface area (A) of the working electrode.
critical care analytes (gases, electrolytes, metabolites) di- When a potential is applied to a working electrode that
rectly in whole blood without need for sample pretreat- will oxidize or reduce a species in the solution phase contact-
ment or dilution. ing the electrode, the electrochemical reaction causes the
• Measurement is rapid and nondestructive. concentration of electroactive species to decrease at the
• Measurement is not affected by sample turbidity (red cells, surface of the electrode (Fig. 17.7), a process termed concen-
lipids); only the analyte present in the water phase of tration polarization. This, in turn, causes a concentration
plasma is measured. gradient of analyte species between the bulk sample solution
• Simultaneous measurement of multiple analytes with an and the surface of the electrode.39 When the bulk solution is
array of sensors in the same blood sample is possible. stirred, the diffusion layer of the analyte grows out from the

Cathodic
VOLTAMMETRY AND/OR AMPEROMETRY il
Voltammetric and amperometric techniques are among the Decomposition
most sensitive and widely applicable of all electroanalytical potential Limiting current
methods.

Basic Concepts i 0
In contrast to potentiometry, voltammetric and amperomet-
ric methods are based on electrolytic electrochemical cells in
which an external voltage is applied to a polarizable working E1/2 – Potential at which 1/2
electrode (measured vs. a suitable reference electrode: Eappl 5 limiting current occurs
Ework 2 Eref ), and the resulting cathodic (for analytical reduc- Anodic
tions) or anodic (for analytical oxidations) current of the cell
is monitored and is proportional to the concentration of the Eappl Ew Eref
analyte present in the test sample. Current flows only if Eappl FIGURE 17.6 Current versus voltage curve (voltammogram)
is greater than a certain voltage (decomposition voltage) obtained for oxidized species (ox) reduced (red) at the surface of
determined by the thermodynamics for a given redox reac- the working electrode, as the Eappl is scanned more negatively
tion of interest (Ox 1 ne2 I Red, which is defined by the and the solution is stirred to yield a steady-state response.

Sample Solution this case); F 5 Faraday’s constant (96,487 coulombs/mol);

Electrode
A 5 electrochemical surface area of the working electrode (in
Ox square centimeters) (assuming a planar electrode geometry);
D 5 diffusion coefficient (in square centimeters per second)
ne
of the electroactive species (Ox in this case);  5 diffusion
layer thickness (in centimeters); and C 5 concentration of
the analyte species (in moles per cubic meter). The D/ term
is often denoted as mo, the mass transfer coefficient of the Ox
Red
species to the surface of the working electrode. Note that Eq.
(17.20) indicates a linear relationship for limiting current
and concentration. The exact same equation applies for
detecting reduced species by an oxidation reaction at the
working electrode. In this case, by convention, the resulting
bulk
anodic current is considered a negative current. As shown in
Cox Fig. 17.6, the potential of the working electrode that corre-
t1 t2 t3 sponds to a current that is exactly one-half the limiting
current is termed the E1/2 value. This value is not dependent
surface
on analyte concentration. The E1/2 is determined by the ther-
Cox modynamics (E0) of the given redox reaction, the solution
conditions (e.g., if protons are involved in reaction, then the
pH will influence the E1/2 value), and any overpotential
caused by slow electron transfer, and so on, at a particular
working electrode surface. The E1/2 values are indicative of a
0 given species undergoing an electrochemical reaction under
Distance specified conditions; hence, the E1/2 values enable distin-
FIGURE 17.7 Concept of electrochemical reaction increasing guishing one electroactive species from another in the same
diffusion layer thickness (concentration polarization) of analytes sample. If the E1/2 values for various species differ significantly
via reduction (red) (or oxidation [ox]) at the surface of the working (e.g., more than 120 mV), then measurements of several limit-
electrode. As time (t) increases, the diffusion layer thickness ing currents in a given voltammogram can yield quantitative
grows quickly to a value determined by the degree of convection results for several different species simultaneously.
in the sample solution.
Electrochemical cells used to carry out voltammetric or
amperometric measurements can involve a two- or three-
electrode configuration. In the two-electrode mode, external
surface of the electrode quickly to a fixed distance, which is voltage is applied between the working electrode and a refer-
controlled by how vigorously the solution is stirred. This dif- ence electrode, and the current is monitored. Because the
fusion layer is termed the Nernst layer and has a finite thick- current must also pass through the reference electrode, such
ness (d) after a relatively short time period (see Fig. 17.7) current flow can potentially alter the surface concentration of
when the solution is moving (convection). Voltammetry carried electroactive species that poise the actual half-cell potential of
out in the presence of convection (by stirring the solution, the reference electrode, changing its value by a concentration
rotating the electrode, flowing solution by electrode, and so polarization process. For example, if an Ag/AgCl reference
on) is called steady-state voltammetry. When the solution is electrode were used in a cell in which a reduction reaction for
not moving, the diffusion layer grows further and further the analyte occurs at the working electrode, then an oxidation
with time (i.e., not constant), creating larger and larger d. values reaction would take place at the surface of the reference
over time. This is termed non–steady-state voltammetry and electrode:
often results in peak currents in i versus Eappl plots for electro-
Ag0 1 Cl2 n AgCl(s) 1 1e2 (17.21)
lytic cells.
In steady-state voltammetry, when the potential of the Hence, the activity and/or concentration of Cl ions near 2

working electrode is scanned past a value that will cause an the surface of the electrode would decrease, which would
electrochemical reaction, the current will rise rapidly and make the potential of the reference electrode more positive
then will plateau, even as Eappl changes further. Fig. 17.6 than its true equilibrium value based on the actual activity of
illustrates such a wave for a hypothetical reduction of an oxi- the Cl2 ion in the reference half-cell, because the Nernst
dized species (Ox) via an n electron reduction to a reduced equation for this half-cell is:
species (Red). When the applied potential is much more
negative than required, the current reaches a limiting value
E Ag AgCl
0
E Ag AgCl (
0.059 log aClsurface
) (17.22)
(termed the limiting current, il). This limiting current is pro-
portional to the concentration of the electroactive species Such concentration polarization of the reference electrode
(Ox in this case) as expressed by the following equation: is prevented by keeping the current density (amperes per
 D square centimeter) low at the reference electrode. This is
i1 nFA   Cox (17.20) achieved in practice by making sure that the area of the work-
 
ing electrode in the electrochemical cell is much smaller than
where i 5 measured current in amperes; n 5 the number the surface area of the reference electrode; hence, the total
of electrons in the electrochemical reaction (reduction in current flow will be limited by this much smaller area, and

current density values for the reference will be very small, as proportional to concentration. Amperometry can be more
desired, to prevent concentration polarization. sensitive than common voltammetric methods because back-
A three-electrode potentiostat is often used to completely ground charging currents, which arise from changing the
eliminate changes in reference electrode half-cell potentials. Eappl as a function of time in voltammetry, do not exist.
In simple terms, the potentiostat applies a voltage to the Hence, when selectivity can be assured at a given Eappl value,
working electrode, which is measured versus a reference elec- amperometry may be preferred to voltammetric methods for
trode via a zero-current potentiometric-type measurement, more sensitive quantitative measurements.
but the current flow is between the working electrode and a
third electrode, called the counter electrode. Thus if reduc- Applications
tion takes place at the working electrode, oxidation would Molecular O2 is capable of undergoing several reduction re-
occur at the counter electrode, but no net reaction would take actions, all with a significant overpotential at solid electrodes,
place at the surface of the reference electrode because no cur- such as Pt, Au, or Ag. For example, the following reaction:
rent flows through this electrode. A potentiostat circuit is
relatively simple to construct using modern operational O2 1 2 H2O 1 4e2 n 4 OH2
(17.23)
amplifiers. (E0 5 10.179V vs. Ag/AgCl; 1 M Cl2)
In voltammetric methods, the Eappl is varying via some
waveform to alter the working electrode potential as a func- exhibits an E1/2 at approximately 20.500 V on a Pt electrode
tion of time and the resulting current measured. The current (vs. an Ag/AgCl reference electrode), with a limiting current
change occurs at the decomposition potential range, which is plateau beginning at approximately 20.600 V. This reaction
best when specific for a given analyte. However, the location is used to monitor the PO2 in blood, which is the basis of the
of the current response as a function of Eappl provides infor- widely used Clark-style amperometric O2 sensor (Fig. 17.8).
mation on the nature of the species present (e.g., E1/2), along This device uses a small area planar Pt electrode as a working
with a concentration-dependent signal. This scan of Eappl can electrode (encased in insulating glass or other material) and
be linear (linear sweep voltammetry) or it can have more an Ag/AgCl reference electrode, typically with a cylindrical
complex shapes that enable greatly enhanced sensitivity to be design (see Fig. 17.8). This two-electrode electrolytic cell is
achieved for monitoring the concentration of a given electro- placed within sensor housing, on which a gas-permeable
active species (e.g., normal pulsed voltammetry, differential membrane (e.g., polypropylene, silicone rubber, Teflon) is
pulse voltammetry, square wave voltammetry).40 When a held at the distal end. The inner working Pt electrode is
dropping Hg electrode is used, such voltammetric methods pressed tightly against the gas-permeable membrane to cre-
are considered polarographic methods of analysis. ate a thin film of internal electrolyte solution (usually buffer
Amperometric methods differ from voltammetry, in that with KCl added). O2 in the sample can permeate across the
Eappl is fixed, generally at a potential value that occurs in membrane and can be reduced in accordance with the pre-
the limiting current plateau region of the voltammogram, ceding electrochemical reaction. An Eappl of 20.650 or 20.700
which simply monitors the resulting current and will be V versus Ag/AgCl (within the limiting current regime) to the

In buffered electrolyte
solution
O2 2H2O 4e→ 4OH
Pt surface

Buffered electrolyte
solution
e
e e e e
Platinum working
electrode
H2O OH

“O” ring
membrane
holder O2

Gas-permeable
membrane
O2 O2 O2 O2 Gas-permeable
membrane

Ag/AgCl cylindrical electrode

FIGURE 17.8 Design of clark-style amperometric oxygen sensor used to monitor partial pressure of
oxygen (PO2) in blood. H2O, Water; Pt, platinum.

Pt working electrode will result in an observed current that is in local dopamine concentrations due to such stimulation
proportional to the PO2 present in the sample (including techniques.
whole blood). In the absence of any O2, the current at Although voltammetric and/or amperometric techniques
this applied voltage under amperometric conditions will be can be applied to detect a wide range of species, the selectivity
near zero. offered for measurements in complex clinical samples—
The outer gas-permeable membrane enables the Clark where many species can be electroactive—is rather limited.
electrode to detect O2 with high selectivity over other easily For example, as stated in the previous discussion relevant to
reduced species that might be present in a given sample (e.g., the Clark O2 sensor, in the absence of the gas-permeable
metal ions, cystine). Only other gas species or highly lipo- membrane, other species that can be reduced at or near the
philic organic species can partition into and pass through same Eappl as O2 would cause significant interference.
such gas-permeable membranes. One type of interference in To expand the range of analytes that can be detected with
clinical samples can be caused by certain anesthesia gases, voltammetric and/or amperometric methods, electrochemi-
such as nitrous oxide, halothane, and isoflurane. These spe- cal techniques can be used as highly sensitive detectors for
cies can also diffuse through the outer membrane of the sen- modern high-performance liquid chromatography systems
sor, can be electrochemically reduced at the Pt electrode, and (see Chapter 19). In liquid chromatography with electro-
can yield a false-positive value for the measurement of PO2.41 chemical detection (LC-EC), eluting solutes are detected by
However, optimized gas-permeable membrane materials and flow-through electrodes (usually carbon or Hg) designed to
appropriate control of the applied potential to the cathode of have extremely low dead volumes (Fig. 17.9). The electrodes
the sensor have greatly reduced this problem in modern in- can be operated in amperometric or voltammetric modes
struments. The outer gas-permeable membranes also help (with high scan speeds), and several electrodes can be oper-
restrict the diffusion of analyte to the inner working elec- ated simultaneously in series or in parallel flow arrangements
trode; hence, the membrane can control the mass transport to gain additional selectivity.45 For example, homocysteine
of analyte (D/ term in Eq. 17.20), such that in the presence can be measured with (1) the addition of reducing agents to
or absence of sample convection, mass transport of O2 to the a serum sample to generate free homocysteine, (2) precipita-
surface of the Pt working electrode is essentially the same. tion of proteins in the sample (with trichloroacetic acid), and
The basic design of the Clark amperometric PO2 sensor (3) separation of the serum components on a reversed-phase
can be used to detect other gas species by altering the applied octadecylsilane high-performance liquid chromatography
voltage to the working electrode. For example, it is possible to column. The eluting homocysteine is detected and measured
detect nitric oxide (NO) with high selectivity using a similar with electrochemical detection via homocysteine oxidation
gas electrode design in which the Pt is polarized at 10.900V to the corresponding mercuric dithiolate complex:
versus Ag/AgCl to oxidize diffusing NO to nitrate at the Pt
2RSH 1 Hg n Hg(RS)2 1 2H1 1 2e2 (17.24)
anode.42 Such NO sensors can be used for a variety of bio-
medically important studies to deduce the amount of NO using a thin-layer Hg/Au amalgam electrode poised at 10.150 V
locally at or near the surface of various NO-producing cells. versus Ag/AgCl.46 Integration of the eluting band for homo-
Beyond amperometric devices, one specialized method of cysteine provides quantitative results, with high selectivity.
detecting trace concentrations of toxic metal ions in clinical Similarly, catechols and catecholamines can be readily de-
samples is anodic stripping voltammetry (ASV). In ASV, a tected in serum by a similar LC-EC method, with eluted
carbon working electrode is used (sometimes further coated catechols oxidized to quinones at a flow-through carbon
with an Hg or Bi films), and the Eappl is first fixed at a negative working electrode poised at potentials typically more than
Eappl voltage so that all metal ions in the solution will be re- 0.200 V versus Ag/AgCl. Furthermore, a host of therapeutic
duced to elemental metals (M0) within the Hg or Bi film and/ drugs can also be quantitated in serum or urine via LC-EC
or on the surface of the carbon. Then, the Eappl is scanned methods.
more positively, and reduced metals deposited in and/or on
the surface of the working electrode are reoxidized, giving a POINTS TO REMEMBER
large anodic current peak proportional to the concentration
of metal ions in the original sample. The potential at which Predominant Types of Electrochemical Sensor
these peaks are observed indicates which metal is present, and Technologies Used in Whole Blood Analyzers
the height of the stripping peak current is directly propor- • Potentiometry: for measurement of pH, PCO2, and electro-
tional to the concentration of the metal ion in the original lytes
sample. Such ASV techniques can be used to detect the total • Amperometry: for measurement of PO2 and as the basis
concentration of lead in whole blood samples, providing a for whole blood glucose and lactate biosensors
rapid screening method for lead exposure and poisoning.43 • Voltammetry: for measurement of Pb21
Another biomedical example of modern voltammetry is a • Conductometry: for measurement of hematocrit
rapid scan cyclic voltammetric technique that has been used
to quantify dopamine in brain tissue of freely moving ani-
mals.44 In this application, oxidation of dopamine to a quinone CONDUCTOMETRY
species at an implanted microcarbon electrode (at approxi-
mately 10.600 V vs. Ag/AgCl) yields peak currents propor- Conductometry is an electrochemical technique used to de-
tional to dopamine concentrations. The electrode can be termine the quantity of an analyte present in a mixture by
used to measure this neurotransmitter in different regions measurement of its effects on the electrical conductivity of
of the brain or in a fixed location. Often, pharmacologic the mixture. It is the measure of the ability of ions in solution
or electrical stimulation can be used to measure the change to carry current under the influence of a potential difference.

Thin-layer EC detector
Sample R O Waste

Working electrode
e
HPLC packed column

Time
FIGURE 17.9 Schematic of liquid chromatography with electrochemical detection (LC-EC) system,
with electrochemical detector monitoring elution of analytes from a high-performance liquid chroma-
tography (HPLC) column by their oxidation or reduction (shown here as example) at a suitable thin-layer
working electrode.

In a conductometric cell, potential is applied between two protein concentrations resulting from dilution of blood with
inert metal electrodes. An alternating potential with a fre- protein-free electrolyte solutions during cardiopulmonary
quency between 100 and 3000 Hz is used to prevent polariza- bypass surgery will result in erroneously low hematocrit
tion of the electrodes. A decrease in solution resistance results values by conductivity. Preanalytical variables, such as insuf-
in an increase in conductance, and more current is passed ficient mixing of the sample, will also lead to errors.50 Hemo-
between the electrodes. The resulting current flow is also al- globin is the preferred analyte to monitor blood loss and the
ternating. The current is directly proportional to solution need for transfusion during trauma and surgery. However,
conductance. Conductance is considered the inverse of resis- electrochemical measurement of hematocrit in conjunction
tance and may be expressed in units of ohm21 (siemens). In with blood gases and electrolytes remains in use mainly
clinical analysis, conductometry is frequently used for mea- because of its simplicity and convenience, despite some
surement of the volume fraction of erythrocytes in whole limitations.
blood (hematocrit) and as the transduction mechanism for Another clinical application of conductance is for elec-
some biosensors, as described below. tronic counting of blood cells in suspension. Termed the
Erythrocytes act as electrical insulators because of their Coulter principle, it relies on the fact that the conductivity of
lipid-based membrane composition. This phenomenon was blood cells is lower than that of a salt solution used as a sus-
used first in the 1940s to measure the volume fraction of pension medium.51 The cell suspension is forced to flow
erythrocytes in whole blood (hematocrit) by conductivity47 through a tiny orifice. Two electrodes are placed on either
and is used today to measure hematocrit on multianalyte side of the orifice, and a constant current is established be-
instruments for clinical analysis. The conductivity of whole tween the electrodes. Each time a cell passes through the ori-
blood depends not only on the volume fraction and shape of fice, resistance increases; this causes a spike in the electrical
the erythrocytes, but also on the conductivity of the sur- potential difference between the electrodes. The pulses are
rounding plasma. An increase in the volume fraction of then amplified and counted.
erythrocytes that are less conductive than the surrounding
plasma leads to a decrease in conductivity shown by the fol- COULOMETRY
lowing relationship48:
a H Coulometry measures the electrical charge passing between
Gb c (17.25)
1 100 H two electrodes in an electrochemical cell. The amount of
charge passing between the electrodes is directly proportional
where Gb 5 conductivity of whole blood; a 5 plasma con- to oxidation or reduction of an electroactive substance at one
ductivity; H 5 percent of hematocrit; and c 5 factor for of the electrodes. The number of coulombs transferred in this
erythrocyte orientation. In practice, plasma conductivity also process is related to the absolute amount of electroactive
contains correction factors for Na1 and K1 concentrations. substance by Faraday’s law:
These cations are usually measured in conjunction with he-
Q5n3N3F (17.26)
matocrit on systems designed for clinical analysis.
Conductivity-based hematocrit measurements have limi- where Q 5 amount of charge passing through the cell (unit:
tations.49 Abnormal protein concentrations will change C 5 coulomb 5 ampere 3 second); n 5 the number of
plasma conductivity and interfere with measurement. Low electrons transferred in the oxidation or reduction reaction;

N 5 the amount of substance reduced or oxidized in moles; Optical Isolator

and F 5 Faraday constant (96,487 coulombs/mol). Fluorescent Sensor Layer
The measurement of current is related to the charge as the A A
BLOOD A A
amount of charge passed per unit time (ampere 5 coulomb A
per second). Coulometry is used in clinical applications for
the determination of Cl2 in serum or plasma and as the
mode of transduction in certain types of biosensors.
Commercial coulometric titrators have been developed Lens
for determination of Cl2. A constant current is applied be-
Plastic
tween an Ag wire (anode) and a Pt wire (cathode). At the Excitation
anode, Ag is oxidized to Ag1. At the cathode, H1 is reduced Light
to H2 gas. At a constant applied current, the number of cou-
lombs passed between the anode and the cathode is directly
proportional to time (coulombs 5 amperes 3 seconds). Emission or
Therefore the absolute number of Ag1 ions produced at the Reflected light
anode may be calculated from the amount of time current
passes through it. In the presence of Cl2, Ag1 ions formed are
precipitated as AgCl(s), and the amount of free Ag1 in solu-
tion is low. When all Cl2 ions have been complexed, a sudden Detector Source
increase in the concentration of Ag1 in solution is noted.
FIGURE 17.10 General design for in vitro optical sensor de-
Excess Ag1 is sensed amperometrically at a second Ag elec-
signed to detect given analyte in blood sample. Polymer film
trode, which is polarized at negative potential. The excess Ag1 contains dye that changes spectral properties in proportion to
is reduced to Ag, producing a current. When this current ex- the amount of analyte in the sample phase. Example shown is
ceeds a certain value, the titration is stopped. The absolute for sensing film that changes luminescence (fluorescence or
number of Cl2 ions present in the sample is calculated from phosphorescence).
the time during which titration with Ag1 was in progress.
Because of the volumetric amount of serum or plasma
sample originally used, it is possible to calculate the concen- point-of-care locations. In such systems, light can be brought
tration of Cl2 in the sample. Coulometric titration is one of to and from the sensing site by optical fibers or simply by ap-
the most accurate electrochemical techniques because the propriate positioning of light sources (light-emitting diodes),
method measures the absolute quantity of electroactive sub- filters, and photodetectors to monitor absorbance (by reflec-
stance in the sample. Coulometry is considered the gold tance), fluorescence, or phosphorescence (Fig. 17.10).
standard for determination of Cl2 in serum or plasma. How-
ever, the method is subject to interference from anions in Basic Concepts
the sample that have affinity for Ag1 more than Cl2 (e.g., Optical sensors devised for PO2 measurements are typically
bromide)52 and is not commonly used in today’s clinical based on immobilization of certain organic dyes (e.g.,
laboratories. pyrene, diphenylphenanthrene, phenanthrene, fluoranthene)
or metal ligand complexes (e.g., ruthenium[II] tris[dipyridine],
Pt and palladium metalloporphyrins) within hydrophobic
OPTICAL CHEMICAL SENSORS polymer films (e.g., silicone rubber) in which O2 is soluble.54
Advances in polymer science, micro and nano particles, The fluorescence or phosphorescence of such species at a
and miniaturization of optical instrumentation (spectrome- given wavelength is often quenched in the presence of para-
ters, light sources, etc.) have contributed to the recent growth magnetic species, including molecular O2. In the case of
in optical-based sensors for clinical applications such as embedded fluorescent dyes, the intensity of the emitted fluo-
immuno- or molecular diagnostics. Optical chemical sensors rescence of such films will decrease in proportion to the PO2
apply optical transduction methods. Absorption, refractive in the sample in contact with the polymer film, in accordance
index, reflectivity, and luminescence (fluorescence and phos- with the Stern-Volmer equation for quenching:
phorescence) are routinely used optical methods in sensors I0
development. For further discussion refer to Chapter 16. An 1 kpO2 (17.27)
I pO2
optode is an optical sensor used in analytical instruments to
measure blood gases electrolytes and metabolites. Optodes where I0 5 fluorescence intensity in the absence of any O2;
have certain advantages over electrodes, including (1) ease IPO2 5 fluorescence intensity at a given PO2; and k 5 quench-
of miniaturization, (2) less noise (no transduction wires), ing constant for the particular fluorophore used.
(3) potential long-term stability using ratiometric-type mea- Hence, a linear relationship exists between the ratio I0/IPO2
surements at multiple wavelengths,53 and (4) they do not and the PO2 in the sample phase. The larger the Stern-Volmer
require a separate reference electrode. These advantages ini- constant, the greater is the degree of quenching for the given
tially promoted the development of optical sensor technol- fluorophore. However, it is important that the quenching
ogy for the design of intravascular blood gas sensors (see the constant is in a range that will yield linear Stern-Volmer be-
section on the In Vivo Sensor), and the same basic sensing havior over the physiologically relevant range of PO2 in
principles have successfully been applied in clinical chemistry blood. For example, if k is too large, then the maximum
instrumentation designed for in vitro measurements on quenching possible will occur over a range of PO2 that is less
whole blood, plasma, or serum samples at laboratory or than physiologic.

Phosphorescence intensity or phosphorescence lifetime within a hydrogel matrix, similar to the pH sensors described
measurements of immobilized metal ligand complexes can earlier. The recognition agent in this case is not usually lipo-
also be used (i.e., binding of O2 decreases excited state life- philic; therefore it must be covalently anchored to the hydro-
times). Sensors based on changes in luminescent lifetime gel, so that it does not leach into the sample phase. The agent
have the inherent advantage of being insensitive to perturba- is designed so that selective cation or anion binding alters the
tions in the optical path length and the amount of active dye absorbance or fluorescence spectrum of the species within the
present in the sensing layer. PO2 sensors based on this tech- hydrogel. Typically, this is achieved by linking ion recognition
nology has been successfully used in single use or multi-use and chromophoric properties within a single organic mole-
blood gas analyzer platforms for point-of-care measurement cule. Such ion sensors have been used successfully in at least
of PO2 and glucose. one commercial blood gas-electrolyte analyzer using an array
Optical pH sensors require immobilization of appropriate of sensors of the generic design similar to that in Fig. 17.10.
pH indicators (e.g., fluorescein, 8-hydroxy-1,2,6-pyrene tri- Optical sensors using fluorescent dyes or polymers cou-
sulfonate, phenol red) within thin layers of hydrophilic poly- pled with boronic acid functional groups have been applied
mers (e.g., hydrogels) because equilibrium access of protons for nonenzymatic glucose sensor development.55 Such sens-
to the indicator is essential. The absorbance or fluorescence ing methods are successfully applied in sensors for both in
of the protonated or deprotonated form of the species can be vitro and in vivo glucose monitoring.
used for sensing purposes.53 One issue with respect to using
immobilized indicators for accurate physiologic pH measure- BIOSENSORS
ments is the effect of ionic strength on the acid dissociation
constant (pKa) of the indicator. Because optical sensors mea- A biosensor is a specific type of chemical sensor consisting of
sure the concentration of protonated or deprotonated dye as a biological recognition element and a physicochemical
an indirect measure of H1 ion activity, variations in ionic transducer, often an electrochemical57,58 or an optical device.
strength of the physiologic sample can influence the accuracy The biological element is capable of recognizing the presence
of the pH measurement. of activity and/or concentration of a specific analyte in
solution. The recognition may be a biocatalytic reaction
Applications (enzyme-based biosensor) or a binding process (affinity-based
Optical sensors suitable for the determination of PCO2 use biosensor) when the recognition element is, for example, an
optical pH transducers (with immobilized indicators) as antibody, DNA segment, or cell receptor. Interaction of the
inner transducers in an arrangement similar to the classic recognition element with a target analyte results in a direct
Severinghaus-style electrochemical sensor design (see Fig. 17.4). measurable change in transducer signal or an indirect change
The addition of bicarbonate salt within the pH sensing hydro- in a solution property locally at the surface of the device, such
gel layer creates the required electrolyte film layer, which varies as formation of a product or consumption of a reactant. The
in pH depending on the PCO2 in equilibrium with the film. The transducer converts the change in solution property into a
optical pH sensor is covered by an outer gas-permeable hydro- quantifiable electrical or optical signal. The mode of trans-
phobic film (e.g., silicone rubber) to prevent proton access, yet duction may be one of several, including electrochemical or
it allows CO2 equilibration with the pH sensing layer. As the optical measurement and measurement of mass or heat. The
PCO2 in the sample increases, the pH of the bicarbonate layer present discussion is limited to biosensors based on electro-
decreases, and the corresponding decrease in the deprotonated chemical and optical modes of transduction because they
form of the indicator (or increase in the protonated form) is constitute most biosensors used for clinical applications.
sensed optically.
Two approaches have been used to sense electrolyte ions POINTS TO REMEMBER
optically in physiologic samples. One method uses many of
the same lipophilic ionophores developed for polymer mem- Biosensors
brane–type ISEs (see Fig. 17.3).12,13 These species are doped • Biosensors measure substances lacking direct electroac-
into thin hydrophobic polymeric films along with a lipophilic tive or optical properties.
pH indicator. In the case of cation ionophores (e.g., valino- • Biosensors consist of a biological recognition element and
mycin for sensing K1), when cations from the sample are a physicochemical (e.g., electrochemical or optical) trans-
extracted by the ionophore into the thin film, the pH indica- ducer.
tor (RH) loses a proton to the sample phase to maintain • Interaction of a biological recognition element with target
charge neutrality within the organic film (yielding R2). This analyte results in either a biocatalytical reaction or a bind-
results in a change in the optical absorption or fluorescence ing process, producing a measurable signal.
spectrum of the polymer layer. If the thickness of the films is • Biosensors are used in most commercial blood glucose
kept at less than 10 mm, equilibrium response times on the meters and continuous glucose monitoring systems.
order of less than 1 minute have been achieved. The main • Sensors for whole blood measurements without a biologi-
limitation of this design is that the pH of the sample phase cal recognition element (e.g., ISEs, optodes) are not con-
also influences the overall extraction equilibrium for ions sidered biosensors.
into the film. Thus simultaneous and independent measure-
ment of sample pH is required, or buffered dilution and/or
pH control of the sample phase is necessary to obtain accu- Enzyme-Based Biosensors With Amperometric
rate measurements of electrolytes. Detection
A second technique used to sense electrolyte ions is im- Enzyme-based biosensors based on electrochemical trans-
mobilization of a cation and/or anion recognition agent ducers, specifically amperometric electrodes, are the most

Platinum i

Glass Reference
electrode

Gas-permeable
membrane
Enzyme O2 O2 O2
layer S O2 P H2O2

Decrease in
Substrate Semipermeable
oxygen within
(e.g., glucose) membrane
enzyme layer
FIGURE 17.11 Illustration of enzyme electrode prepared using oxidase enzyme immobilized at the sur-
face of an amperometric partial pressure of oxygen (PO2) sensor. Increase in substrate concentration (S)
reduces the amount of oxygen present at the surface of the sensor. H2O2, Hydrogen peroxide.

commonly used for clinical analyses and the most frequently In practice, a sufficiently high voltage (overpotential)
cited in the literature.58 Blood glucose meters, for home must be applied to the platinum anode to drive the oxidation
monitoring or for hospital use, are based on enzyme biosen- of the H2O2. An applied voltage of 10.7 V or greater (relative
sors with amperometric detection and glucose sensors in to Ag/AgCl) is typically used. Fig. 17.12 illustrates this basic
single-use formats. Clark and Lyons developed the first am- H2O2 detection design, which is suitable for use in devising
perometric biosensor; it was used to measure glucose in clinically useful sensors for glucose, but also for a host of
blood and was based on immobilizing glucose oxidase on other substrates for which suitable oxidase enzymes generate
the surface of an amperometric PO2 sensor.59 A solution of H2O2. For example, immobilization of lactate oxidase (in-
glucose oxidase was physically entrapped between the gas- stead of glucose oxidase), in the configuration described for
permeable membrane of the PO2 electrode and an outer glucose sensors, produces a biosensor for the measurement of
semipermeable membrane (Fig. 17.11; see general design). lactate in blood, plasma, or serum samples.
The outer membrane was of a low-molecular-weight cutoff Immobilization of enzymes in the early biosensors was
to allow the substrate (glucose) and O2 from the sample to a simple entrapment method behind a membrane of low-
pass, but not proteins and other macromolecules. In this molecular-weight cutoff; this approach is still used in some
way, enzymes could be concentrated at the sensor’s surface. commercial applications. Many other schemes for enzyme
Oxidation of glucose, catalyzed by glucose oxidase as follows: immobilization for biosensor development have been sug-
gested.60 The most common are cross-linking of the enzyme

Glucose 1 O2 glucose
 → gluconic acid 1 H2 O 2
oxidase
(17.28) with or without an inert protein (e.g., bovine serum albu-
min), using glutaraldehyde, simple adsorption of enzyme to
consumes O2 near the surface of the sensor. The rate of de- electrode surfaces, and covalent bonding of enzymes to in-
crease in PO2 is a function of the glucose concentration and soluble carriers (e.g., nylon or glass). Another immobiliza-
is monitored by the PO2 electrode. A steady-state PO2 can be tion technique involves bulk modification of an electrode
achieved at the surface in a short period of time, yielding a material, mixing enzymes with carbon paste or inks, which
steady-state current value that decreases as a function of glu- serve as both the enzyme immobilization matrix and the
cose concentration in the sample. For additional information electroactive surface.61
on glucose measurement, refer to Chapter 35. One of the first biosensor-based systems for the measure-
If the polarizing voltage of the PO2 electrode is reversed, ment of glucose in blood was commercialized by Yellow
making the Pt electrode positive (anode) relative to the Ag/ Springs Instruments (YSI), Inc. in 1975, and used the am-
AgCl reference electrode, and if the gas-permeable mem- perometric detection of H2O2 as the measurement principle
brane is replaced with a low-molecular-weight cut-off mem- (see Fig. 17.12). Dependence of the measured glucose value
brane and immobilizing enzyme on the platinum electrode, on O2 concentration in the sample was a problem because
it is possible to oxidize the hydrogen peroxide (H2O2) pro- significantly less than the stoichiometric amount of dissolved
duced by the glucose oxidase as follows: O2 is present in blood to support the glucose oxidase reaction
and to produce a linear relationship of signal with glucose
H2O2 n 2H1 1 O2 1 2e2 (17.29)
concentration. This is especially true at high concentrations
The steady-state current produced is now directly propor- of glucose found in samples from patients with diabetes
tional to the concentration of glucose in the sample. (more than 500 mg/dL; 27.8 mmol/L). In the case of the YSI

0.7V Electrode

MOx GOOx Product

i CurrentK[glucose]

Ag/AgCl MRed GORed Glucose

Pt anode e
FIGURE 17.13 The use of an electroactive mediator in the de-
sign of an amperometric enzyme electrode. The mediator accepts
electrons directly from the enzyme and is oxidized at the surface
of the working electrode, creating more oxidized mediator to
continue this process. (From D’Orazio P. In: Lewendrowski K, editor.
Inner and Clinical chemistry: laboratory management and clinical correla-
Immobilized outer tions. Philadelphia: Lippincott, Williams & Wilkins; 2002:464.)
enzyme membranes
A
Electron acceptors other than O2 can serve as mediators in
the glucose oxidase reaction and completely eliminate any
dependence of the amperometric response on the O2 concen-
tration of the sample. The mediator, which is usually co-
Pt anode immobilized with the enzyme, transports electrons to the
anode surface, where it is reoxidized, resulting in a cyclic reac-
H2O2 2e → O2 2H tion mechanism (Fig. 17.13). Mediators with electron trans-
Low MW
cut-off fer kinetics (little or no overpotential) more favorable than
membrane that of O2 allow operation of the sensor at lower applied po-
H2O2 ← O2 Gluc Glucose
Outer oxidase tentials (10.2 V vs. Ag/AgCl or lower) than those that are
membrane typically used for the oxidation of H2O2. This approach not
O2 O2 Gluc only eliminates dependency of the reaction rate on O2, it also
Gluc rbc Gluc rbc serves to reduce the contribution from oxidizable interfering
rbc substances (e.g., uric acid, ascorbic acid, acetaminophen) to
rbc Gluc the sensor response. Examples of mediators that have been
B Gluc
used include quinones and conductive organic salts, such as
FIGURE 17.12 (A) Design of amperometric enzyme electrode
based on anodic detection of hydrogen peroxide (H2O2) gener-
tetrathiafulvalene-tetracyanoquinodimethane.65,66 Ferricya-
ated from oxidase enzymatic reaction (e.g., glucose oxidase). (B) nide, ferrocene, or redox osmium polymers have also been
Expanded view of the sensing surface shows the different mem- used,67 including commercial applications in devices for
branes and electrochemical processes that yield the anodic cur- home blood glucose monitoring. Dimethylferrocene is im-
rent proportional to the substrate concentration in the sample. pregnated into a graphite electrode to which glucose oxidase
Ag/AgCl, Silver-silver chloride; K, proportionality constant; MW, has been immobilized. Reduced glucose oxidase from the
molecular weight; Pt, platinum; rbc, red blood cells. (From Mey- enzymatic reaction is reoxidized by the electrochemically
erhoff M. New in vitro analytical approaches for clinical chemistry generated ferricinium ion. Current produced during this cy-
measurements in critical care. Clin Chem 1990;36:1570.) cling mechanism is proportional to the concentration of
glucose in the blood sample.
Another technique used to decrease interferences from eas-
system, sample and calibration solutions are diluted at least ily oxidized species in a blood sample when traditional H2O2
1:10 in buffer (depending on the model), which is in equilib- electrochemical detection is used is to employ selectively
rium with atmospheric PO2 and fixes the O2 concentration in permeable membranes in proximity to the electrode surface
the calibrator and sample at a constant value. that allow transport of H2O2 to the electrode surface, but
The problem of O2 limitations of biosensors based on reject the interfering substances based on size or charge
oxidase enzymes has been addressed by designing semiper- exclusion (see Fig. 17.12B). An example is polymeric mem-
meable membranes that restrict diffusion of the primary brane from cellulose acetate or a polyether sulfone derivative,
analyte (substrate) to the enzyme layer, which avoids satura- which is used in many commercial amperometric biosensors.68
tion of the enzyme and keeps the ratio of O2 to analyte always Electropolymerized films, such as poly(phenylenediamine)
in excess of 1. This extends the linearity of response to analyte formed in situ, are also used, to reject interfering substances
concentrations substantially higher than the Km of the enzyme based on size.69 Another approach used in a commercial
and reduces the signal dependence on O2. Outer track–etched application involves using a second correcting electrode, iden-
polycarbonate or polyethylene terephthalate membranes are tical to the working electrode, but without an enzyme that is
commonly used,62 as are membranes of poly(vinyl Cl), poly- sensitive only to the presence of oxidizable interfering sub-
urethanes, and silicone emulsions.63 Another approach has stances. The resulting differential signal is proportional to the
been to use an O2-rich electrode material as a reservoir of O2 concentration of analyte.
to support the bioreaction. A fluorocarbon (Kel-F Oil) has A novel approach used for elimination of electroactive
been used to formulate a carbon paste electrode to act as a interfering substances in a commercially available glucose
source of O2 and as the working electrode.64 sensor is to directly “wire” the redox center of the enzyme

glucose oxidase to a metallic, amperometric electrode using mV

an osmium (III/IV)-based redox hydrogel.70 Osmium sites
effectively serve as mediators and can accept electrons directly
from the entrapped enzyme, without the need for O2. This
Reference
approach allows the operating potential of the electrode to be electrode
dramatically lowered to 10.2 V versus the saturated calomel
reference electrode, where currents resulting from electro-
Internal electrolyte
oxidation of ascorbate, urate, acetaminophen, and l-cysteine
are negligible.
Substitution of other oxoreductase enzymes for glucose Ammonium selective
oxidase allows amperometric biosensors for other substrates polymeric membrane
of clinical interest to be constructed. Practical sensors with (nonactin)
commercial application in critical care analyzers for blood
lactate have been realized.71 By using the multiple enzyme Immobilized
cascade shown in the reactions discussed here, an ampero- urease
metric biosensor for creatinine is also possible. Electrochem-
ical oxidation of H2O2 is the detection mechanism:
Nonactin
Creatinine 1 H 2 O creatinine
  → Creatine
amidohydrolase
(17.30) NH4 K

Creatinine 1 H 2 O creatine
  → Sarcosine 1 Urea (17.31)
amidinohydrolase
Crosslinked urease
2NH K
enzyme layer 4 CO2 urea
Sarcosine 1 H 2 O 1 O 2 → Glycine
sarcosine oxidase
(17.32)

1 formaldehyde 1 H2 O2 rbc rbc
K urea
urea K
This three-enzyme scheme suffers interference from en- rbc
rbc rbc
dogenous creatine in the sample, requiring correction. Low urea
concentrations of creatinine found in blood (1.13 mg/dL or FIGURE 17.14 Potentiometric enzyme electrode for determina-
less; 100 mmol/L or less) must be measured in the presence of tion of blood urea, based on urease enzyme immobilized on the
oxidizable interfering substances, which are sometimes pres- surface of an ammonium ion–selective polymeric membrane
ent at higher concentrations than the analyte.72 Special elec- electrode. CO2, Carbon dioxide; K1, potassium; NH4+, ammonium;
troactive layers within the biosensor have been proposed to rbc, red blood cells.
remove redox-active interfering substances.73 Because the
useful life of the creatinine or lactate biosensors requires
enzyme(s) to retain activity, reusable commercial biosensors in the sample. The response may be steady state or transient.
for creatinine or lactate typically have a short (few days) use- Typically, correction for background K1 is required, be-
ful life, but improvements in enzyme immobilization meth- cause the nonactin ionophore has limited selectivity for
ods and/or use of stabilizers and/or activators within cali- NH41 over K1 (KNH4/K 5 0.1). Potassium is measured si-
brating reagents have yielded creatinine or lactate sensor multaneously with urea and is used to correct the output
devices with much longer lifetimes (3 weeks of continuous of the urea sensor using the Nikolsky-Eisenman equation
use after ambient storage for 6 months). Such improvements (Eq. 17.11).
in stability and ability to measure directly in whole blood has The approach already described for measurement of urea
resulted in commercial applications of these biosensors for using an enzyme-based potentiometric biosensor assumes
point of care, specifically, at emergency departments for basic that the turnover of urea to NH41 at steady state provides a
metabolic panel testing. The importance of developing cre- constant ratio of NH41 ions to urea, independent of concen-
atinine sensors and progress to date in this field have recently tration. This is rarely the case, especially at higher substrate
been summarized in detail by Lad and colleagues.74 concentrations, which results in a nonlinear sensor response.
The linearity of the sensor is also limited by the fact that hy-
Enzyme-Based Biosensors With Potentiometric drolysis of urea produces a local alkaline pH in the vicinity of
and Conductometric Detection the NH41 sensing membrane, partially converting NH41 to
ISEs can be used as transducers in potentiometric biosensors. NH3 (pKa 5 9.3). Ammonia is not sensed by the ISE. The
An example is a biosensor for urea (BUN) based on a polymer degree of nonlinearity may be reduced by the placement of a
membrane ISE (poly[vinyl Cl]) for NH41 ion (Fig. 17.14).75 semipermeable membrane between the enzyme and the sam-
The enzyme urease is immobilized at the surface of the NH41 ple to restrict diffusion of urea to the immobilized enzyme
selective ISE based on the antibiotic nonactin (see structure of layer. Several commercial analyzers were introduced recently
ionophore in Fig. 17.3) and catalyzes the hydrolysis of urea to to measure urea directly in whole blood for point-of-care ap-
NH3 and CO2: plications using potentiometric biosensor approach.
Urea urease
→ 2NH3 1 CO2 (17.33) A change in solution conductivity has also been used as
a transduction mechanism in enzyme-based biosensors.
The protonated form of ammonia (NH3), NH41 is sensed Examples include the measurement of glucose, creatinine,
by the ISE. The signal generated by the NH41 produced is and acetaminophen using interdigitated electrodes.76 There
proportional to the logarithm of the concentration of urea are few practical applications of conductometric biosensors

because of the variable ionic background of clinical samples sample matrix. A pH-sensitive indicator may also be used to
and the requirement to measure small conductivity changes follow enzymatic reactions that produce ammonia (e.g., ure-
in media of high ionic strength. A commercial system for the ase action on urea).
measurement of urea in serum, plasma, and urine is a BUN
analyzer (Beckman-Coulter, Brea, California) based on the Affinity Sensors
enzyme urease.77 Dissolution of products to NH41 and Affinity sensors are a special class of biosensors in which the
HCO32 produces a change in sample conductivity. The initial immobilized biological recognition element is a binding pro-
rate of change in conductivity is measured to compensate for tein, antibody (immunosensors), or oligonucleotide (e.g.,
the background conductivity of the sample. This approach is DNA, aptamers, covered in more detail in the following) with
limited to the measurement of analytes at relatively high con- high binding affinity and high specificity toward a clinically
centrations because of small changes in conductivity pro- important analyte and/or partner. Such sensors are being
duced by low concentrations of analyte. developed as alternatives to conventional binding assays to
enhance the speed and convenience of a wide range of assays
Enzyme-Based Biosensors With Optical Detection that normally would be run on large, sophisticated instru-
Optical sensors with immobilized enzymes and indicator ments in a central laboratory. Affinity sensors may be more
dyes have been developed for the measurement of glucose easily adapted than traditional assays to systems developed
and other substrates of clinical interest.78 These biosensors for point-of-care testing for infectious disease, cardiac mark-
are based on optical detection chemistries for pH and O2, ers, or other cases where speed and ease of use are required.
described earlier in this chapter, and rely on absorbance and/ Ideally, direct binding of the immobilized species with its
or reflectance, fluorescence, and luminescence as modes of target in a clinical sample should yield a sensor signal pro-
detection. Enzyme immobilization methods resemble those portional to the concentration of the analyte. However, direct
used to construct electrochemical biosensors, including phys- sensing (without use of exogenous labels and/or tracers) of
ical entrapment or encapsulation in a gel matrix, physical the binding events at analyte concentrations that would cover
adsorption onto substrates, and covalent binding or absorp- the full range for clinical applications is very difficult to
tion on an insoluble support. Using an example based on an achieve. Furthermore, high affinity of such binding reactions,
optode for PO2, a sensitive indicator is co-immobilized with which are required to achieve optimal sensitivity, also limits
an oxidase enzyme at the end of a fiber optic probe. The the reversibility of such devices (slow reverse rate constant).
probe is used to monitor fluorescence of the indicator. Unlike ISEs, O2 sensors, and many of the enzyme-based bio-
Quenching of fluorescence of the indicator by O2 is followed. sensors described previously, affinity sensors based on elec-
A decrease in PO2 resulting from a reaction catalyzed by the trochemical, optical, or other transduction modes are typically
enzyme will result in less quenching of the indicator and a single-use devices. For repeated multiuse applications, some
fluorescent signal directly proportional to the concentration type of regeneration step (pH change, temperature change, and
of the substrate. In an example of an optical biosensor probe so on) to dissociate the tight binding between the recognition
for glucose, an O2-sensitive cationic dye, Ru(phen)321, is im- element and the target is required. An example of a thermally
mobilized along with glucose oxidase on the surface of an reversible immunosensor, demonstrated to retain activity and
optical fiber.79 A decrease in PO2 arising from the enzyme- specificity for up to 30 assays, has recently been described.83 The
catalyzed oxidation of glucose results in an increase in lumi- sensor consists of an antibody conjugated to a polymer, which
nescence intensity of the ruthenium tris(phenanthrene). undergoes a reversible phase transition in response to tempera-
Similar optical biosensors have been prepared for many ture. Altering temperature produces a change in orientation of
other analytes. For example, a cholesterol optical biosensor the conjugated polymer and affinity between the conjugate and
has been devised based on fluorescence quenching of an O2- a target antigen, resulting in a reversible antigen–antibody
sensitive dye that is coupled to the consumption of O2 result- binding reaction.
ing from the enzyme-catalyzed oxidation of cholesterol by Most affinity-type sensors appearing in the literature with
the enzyme cholesterol oxidase.80 Serum bilirubin has been a promise of clinical usefulness are based on labeled reagents,
detected using bilirubin oxidase, co-immobilized with a ru- such as enzymes, fluorophores, and electrochemical tags, and
thenium dye, on an optical fiber.81 The bilirubin sensor was hence, they function more like traditional binding and/or
reported to exhibit a lower detection limit of 0.006 mg/dL immunoassays, except that one recognition element is im-
(0.1 mmol/L), a linear range up to 17.6 mg/dL (300 mmol/L), mobilized on the surface of a suitable electrode or another
and a typical reproducibility of 3% (coefficient of variation), type of transducer.84–86 For example, electrochemical O2 sen-
which is adequate for clinical application. sors have been used to carry out heterogeneous enzyme im-
The pH change resulting from enzyme-catalyzed reactions munoassays (sandwich or competitive type), using catalase as
has also been measured optically. The indicator dye fluores- a labeling enzyme (catalyzes H2O2 n 2H1 and O2) and im-
cein is often used as a pH-sensitive indicator to construct mobilizing capture antibodies on the outer surface of the
such sensors. The protonated form of fluorescein does not gas-permeable membrane. After binding equilibration and
fluoresce, but the conjugate base strongly fluoresces at 530 washing steps, the amount of bound enzyme is detected by
nm, when excited at 490 nm. Using glucose oxidase as the adding the substrate and following the increase in current
enzyme, a pH optode has been used to follow the formation generation caused by local production of O2 near the surface
of gluconic acid.82 A disadvantage of optical sensors based on of the sensor.
pH changes is that they are strongly dependent on the pH The number of research reports related to affinity bio-
and buffer capacity of the sample. Moreover, the working sensors continues to increase, with potential application for
range of the sensor is determined by the pKa of the indicator, detection of markers of cardiac disease, cancer, and autoim-
6.8 to 7.2 for fluorescein, depending on ionic strength of the mune disease.87 Affinity sensors for DNA analysis are covered

in more detail in the following. Commercialization has lagged washing steps are required. This method was used to detect
behind research output, and movement of affinity biosensors prostate-specific antigen (PSA) and human chorionic go-
from the research laboratory to the clinical laboratory has nadotropin at nanogram per milliliters concentrations in
been slow. Some commercial examples of immunosensors do undiluted plasma and whole blood. A number of other di-
exist, primarily in the unit-use, disposable format designed rect-reading affinity sensors have been proposed, based on
for point-of-care testing. One successful commercial example electrochemical (including capacitance and impedance
is a cartridge-based device for cardiac troponin I on the changes), optical, thermal, mass, and acoustic detection
i-STAT handheld analyzer (Abbott Point of Care, Abbott methods.84–86,90,91 Direct affinity sensors eliminate the need
Laboratories, Abbott Park, Illinois).88 This sensor uses a sand- for labeled reagents because the binding reaction results in a
wich immunoassay format with electrochemical detection. A change in a property that may be monitored directly. Of
capture antibody is immobilized on a Au electrode for recog- these, few have adequate specificity to be used in complex
nition and capture of troponin in the blood sample. A second clinical samples because of significant signals arising from
reporter antibody is labeled with alkaline phosphatase and nonspecific binding. However, one report has suggested that
binds with surface-captured troponin antigen. Following in- a-fetoprotein can be detected reliably in serum samples via a
cubation and washing steps, the substrate p-aminophenyl quartz crystal microbalance mass detector, which possesses
phosphate is introduced, and the product of the enzymatic immobilized anti–a-fetoprotein antibodies on the surface of
reaction (p-aminophenol) is detected amperometrically by the quartz crystal transducer.92 Increasing concentrations of
oxidation. Magnitude of the oxidative current is proportional analyte in the sample yield increased binding to the surface,
to concentration of troponin in the sample. changing the mass loaded because of the immunologic reac-
The basic advantage of immobilizing affinity reagents on tion. Incubation times as low as 20 minutes are required to
the surfaces of electrodes and optical sensing devices is some- achieve results that compare favorably with those of a con-
what diminished when separate washing steps are required to ventional (radio-)immunoassay method for serum samples.
remove unbound label species. As discussed previously, true Direct immunosensors for sensitive assays of tumor markers
affinity biosensors should yield analytically useful responses have been reported. Examples include an electrochemical
in the presence of undiluted physiologic samples, without the immunosensor for PSA with an enhanced lower limit of
need for discrete incubation and washing steps. One example detection, based on an alternating current impedance mea-
of an electrochemical-based immunosensor method that surement,93 and an assay for carcinoma antigen-125 using
partially achieves this goal is a technique termed nonsepara- a quartz crystal microbalance.94 The former uses a control
tion electrochemical enzyme immunoassay.89 The basic con- sensor with an immobilized immunoglobulin-G antibody
cept is illustrated in Fig. 17.15. As indicated, no separation or instead of anti-PSA to subtract out effects from nonspecific
binding to achieve a limit of detection of 1 pg/mL toward PSA.

Microporous nylon Affinity Sensors for DNA Analysis Using

membrane Fluorescent Labels
E Affinity-type sensors based on oligonucleotide binding rep-
resent perhaps the most promising application of affinity
E sensors to clinical chemistry. Fluorescent labels were the first
to be used in an early commercial biosensor for DNA analy-
Analyte E sis, introduced in 1996 (GeneChip, Affymetrix, Thermo
E E fisher). High-density oligonucleotide arrays are formed on
glass substrates. Target DNA is isolated and amplified using
the polymerase chain reaction (PCR) while a fluorescent la-
bel is incorporated. The sample is incubated on the array, and
detection of hybridization of labeled DNA to its complemen-
Gold
tary strands takes place by monitoring light emitted by the
Antibody
coating Add substrate to back to fluorescent labels. The primary application of the device has
initiate enzymatic reaction been for DNA sequencing, so the focus has been primarily
on increasing the density of the DNA arrays. Applications of
E the device continue to expand with a variety of arrays devel-
oped for gene expression and whole genomic analysis related
Substrate to (1) cancer, (2) inflammatory disease, (3) infectious disease,
e Product
and (4) diabetes. Another commercial example of affinity
E sensors for DNA analysis using fluorescent labels is the
e E BeadArray technology (Illumina, San Diego, California).95,96
Silica microbeads with covalently attached, single-stranded
DNA probes are randomly assembled on a fiberoptic bundle
or planar silica surface. Each bead carries hundreds of thou-
FIGURE 17.15 Principle of nonseparation electrochemical en-
zyme immunoassay concept, configured in sandwich immunoassay
sands of copies of a gene-specific probe sequence. Due to the
mode to detect a given protein analyte. Enzyme label on reporter high-density packing of beads, with a possibility of approxi-
antibody generates an electroactive product that is detected at the mately 50,000 beads on a 1.4-mm diameter array, a large
surface of the porous gold electrode when the substrate for the number of probe sequences may be investigated in parallel,
enzyme is added to the back of the membrane. and multiple copies of each probe sequence are possible,

which increases measurement precision. Fluorescently la- (Ru[NH3]6)31 is reduced at the electrode surface, and the
beled target sequences in the sample to be analyzed hybridize coulometric signal is proportional to the concentration of
with their complementary probe sequences. Arrays are target DNA.98 A commercial example of electrochemical
scanned in a BeadArray reader for image analysis and data DNA sensing, along with AuNP probes without need for PCR
extraction. This versatile platform has been adapted for vari- amplification, is the Verigene system (Nanosphere, Luminex),
ous genetic analyses, such as single nucleotide polymorphism which is capable of detecting single-nucleotide polymor-
genotyping, DNA methylation detection, and gene expres- phisms related to common genetic disorders, such as (1)
sion profiling.97 thrombophilia, (2) alterations of folate metabolism, (3) cystic
fibrosis, and (4) hemochromatosis. Another commercially
Affinity Sensors for DNA Analysis Using available, electrochemically based platform for nucleic acid
Electrochemical Labels detection without the need for sample purification or target
DNA sensors in which a segment of DNA complementary to amplification is from GeneFluidics (Irwindale, California).
a target strand is immobilized on a suitable electrochemical The system uses a sensor array chip consisting of 16 na-
sensor have been demonstrated. These devices operate in noscale Au electrodes, modified with thiol self-assembled
direct (based on electrochemical oxidation of guanine in monolayers, optimized for biomolecule immobilization.99
target DNA; Fig. 17.16A) or indirect (with exogenous electro- Horseradish peroxidase is the preferred target label because
chemical markers and/or labels, see later and Fig. 17.16B) of its fast electron transfer kinetics, when used with ampero-
transduction modes. Although most of the proposed electro- metric detection.
chemical DNA biosensors require amplification methods, Another example of an electrochemical “gene” sensor ar-
such as PCR, to multiply small amounts of DNA into mea- ray uses electrochemical probes that are selectively inserted
surable quantities, some are sensitive enough to eliminate the into hybridized DNA duplexes. In one approach, after the
need for target amplification. Nanotechnology has been pro- immobilized capture of oligo anchored to the electrode sur-
posed, in the indirect format, for signal amplification. For face is allowed to bind the target sequence, hybridization is
example, a capture probe DNA is immobilized on an Au detected by exposing the surface of the electrode to an exog-
electrode. Reporter probes with electrostatically bound ruthe- enous electroactive species (Co[III]tris-phenanthroline, ru-
nium complexes (Ru[NH3]6)31 are loaded onto Au nanopar- thenium complexes, and so on) that intercalates within the
ticles (AuNP) and are capable of hybridizing with one of duplex, but not to single-stranded DNA. After unbound elec-
two sequences on target DNA. The other sequence on the troactive species are removed by washing, the presence of
target DNA is capable of hybridizing with the immobilized hybridization is readily detected by voltammetry, scanning
capture probe. Hybridization events on the electrode surface the potential of the underlying electrode to oxidize or reduce
bring multiple reporter probes for each AuNP. Electroactive any intercalated electroactive species, with the current

Capture probe Sample Oxidation of guanine on target DNA

T
C
A I A T T I C C T G G
AA
Electrode

C
G
G G G
A I A T T I C C

T e
C
T A A CG
A I A T T I C C G
A

Reduction/oxidation of
Capture probe Sample redox intercalation species
T Ox
C Ox
A I A T T I C C T Ox Ox Ox Ox
AA
Ox
Electrode

C
G e
G Ox Ox Ox Ox
A I A T T I C C
T
C
T A A CG
A I A T T I C C G Red
B
FIGURE 17.16 Examples of DNA biosensor configurations. A, Direct electro-oxidation (Ox) detection
of guanosine bases in target DNA after hybridization with immobilized capture probe on electrode
surface. B, Electrochemical detection of hybridization using exogenous redox (Red) species that inter-
calates into hybridized complex between the immobilized capture DNA probe and target DNA.

detected being proportional to the number of duplex DNA In the absence of thrombin, the aptamer chain is flexible, and
species on the surface of the electrode. the MB label is close to the electrode surface for high electron
transfer efficiency. Upon binding to the thrombin target, the
Affinity Sensors Based on Aptamers (Aptasensors) folding of the aptamer moves the label away from the elec-
Aptamers have been explored as versatile recognition ele- trode surface, inhibiting electron transfer. A sensitivity down
ments for a variety of biosensing applications, including to 6.4-nmol thrombin was demonstrated. The opposite
small molecules, proteins, and cells.100 Aptamers are synthetic transduction approach (in which target binding by the ap-
single-stranded nucleic acids capable of folding into three- tamer brings an electroactive label closer to an electrode
dimensional structures to selectively bind target molecules. surface) was demonstrated as a sensor for platelet-derived
In practice, aptamers may be generated and selected in vitro to growth factor (PDGF) in blood serum.108 In this case, the MB
bind targets for which antibodies and other protein receptors label was attached at one end of the PDGF aptamer, and in
are not easily obtained, using a process known as systematic the absence of a target, the label was relatively far from the
evolution of ligands by exponential enrichment (SELEX).100 electrode surface. Binding of the PDGF target brought the
During the SELEX process, a large, random DNA or RNA MB label close to the electrode surface in a stable configura-
library goes through an iterative process of selective binding tion, enhancing an electron transfer. Detection of PDGF
toward a target analyte. After separation of bound and un- down to 50 pmol was shown.
bound DNA, the binding DNA is amplified using PCR for a Using a similar concept as the thrombin sensors described
subsequent round of selection and isolation of the optimum above, electrochemical aptamer-based sensors were devel-
binding segment. This method, by which aptamers are se- oped for real-time, continuous, and multi-hour tracking of
lected against their targets, makes them inherently suited to four drugs (doxorubicin, kanamycin, gentamicin, and tobra-
displacement assays because of greatly reduced affinity for mycin) in the bloodstream. These sensors were able to
the labeled form of a target. Another inherent advantage of quickly respond (3 seconds) to different concentrations of
aptamer-based affinity sensors is reduced nonspecific bind- these drugs following an injection into the bloodstream and
ing because of the conformational change of the aptamer recovered to baseline response after clearing of the drug from
during the binding event, making the aptamer less suscepti- blood.109 Square wave voltammetry at high frequencies have
ble to recognition of many potential interfering substances in been employed to achieve fast response. Such reusable fast
a complex sample matrix (e.g., blood serum).101 The same responding aptamer-based sensors can provide valuable in-
conformational change may optimize performance of a la- sight to pharmacokinetic information including feedback-
beled target following a binding event, for example, by alter- controlled drug delivery. Electrochemical aptamer sensors
ing the local environment of a fluorescent label or changing are growing rapidly in the scientific literature for different
the position of an electrochemical label with respect to a applications that are traditionally not feasible with ISE, enzyme,
sensing electrode. Although few aptamer-based diagnostic or antibody-based sensors.110 Examples include calibration
products have been commercialized at present, reported free sensors for phenylalanine concentrations, sensors for
practical applications for aptamer-based affinity sensors in insulin monitoring, cocaine in undiluted blood, sensors for
clinical chemistry are increasing in the literature. toxins in food, etc.111–115
Aptamers have been demonstrated to function in biosen-
sors using various transduction methods, including optical POINTS TO REMEMBER
sensing (using fluorescently labeled aptamers),102 and acous-
Affinity Biosensors
tic, mass (cantilever-based), and electrochemical sensing,
• Affinity biosensors are a specific type of biosensor in
which use, for example, aptamer probes labeled with a redox
which an immobilized recognition element exhibits a high
species (e.g., Ferrocene or Methylene Blue).103,104 Many trans-
binding constant for the analyte of interest.
duction modes, such as fluorescence, have a background sig-
• Examples of recognition elements are antibodies and oli-
nal in a complex sample matrix (e.g., blood serum), which
gonucleotides (DNA segments).
limits the sensitivity of the method. One transduction mode,
• Affinity biosensor binding reactions are typically irrevers-
demonstrated using an aptasensor, to reduce background
ible and best suited to single-use sensors.
fluorescence in a complex sample matrix is time-resolved
• Most affinity biosensors for clinical application use indirect
fluorescence, in which the fluorescence lifetime of the detec-
modes of transduction, based on reagent label such as
tion signal is differentiated from nonspecific fluorescence
enzymes, fluorescent, and electrochemical labels.
background in the sample matrix. Lanthanides, such as euro-
pium ions, are attractive candidates as fluorescent labels be-
cause of their long fluorescent lifetimes (up to 1 ms).105 As is
the case with most affinity-based biosensors, electrochemical CHEMICAL SENSORS BASED
sensing for aptasensors may still be the preferred transduc- ON NANOTECHNOLOGY
tion mode because redox labels may be chosen to operate at
an applied potential away from potentially interfering elec- Nanotechnology is defined as the study of the synthesis,
troactive species in the sample matrix. By taking advantage of properties, and application of structures and materials having
the conformational change of the aptamer during a binding at least one critical dimension in the range of 1 to 100 nm.116,117
event, it changes the relative proximity of an electrochemical for additional discussion on microfibrication and microflu-
label to an electrode surface.103,106 Using thrombin as a model idics, refer to Chapter 27. Structures such as nanowires,
analyte and methylene blue (MB) as an electroactive label, an nanoparticles, quantum dots, and carbon nanotubes offer
aptasensor was constructed by immobilizing a MB-labeled, unique electrical, optical, and magnetic properties that can
thrombin-binding aptamer to a thiol modified Au electrode.107 be exploited for chemical sensing. The large surface area

available on nanostructures for immobilization of labels and

biological recognition elements offers the potential for high
IN VIVO AND MINIMALLY INVASIVE SENSORS
signal amplification by increasing the signal-to-noise ratio. Progress has been made in the development of miniaturized,
Nanomaterials and their applications, particularly for the devel- implantable electrochemical and optical sensors that can be
opment of sensors for cancer biomarkers with improved lower used for in vivo, real-time monitoring of clinically important
limits of detection, have recently been reviewed.117,118 Develop- species. Unfortunately, the biological response toward such
ments in the field of DNA nanodevices have resulted in com- sensors (e.g., clotting) can have a dramatic impact on the
plex molecular detection and amplification schemes, which analytical accuracy of such indwelling probes129–132 and has
may outperform PCR in terms of sensitivity and ease of use.119 prevented their widespread use in clinical practice. However,
The flexibility and the unique shape of carbon nanotubes progress is being made toward improving the reliability of in
and the presence of reactive groups on their surface has led to vivo measurements, and some commercial products for sub-
their being used as biocatalytic and affinity biosensors.120,121 cutaneous CGM are already on the market (see later).
Direct electron transfer between immobilized glucose oxi- Analytical sensors that can be implanted within human
dase and carbon nanotubes allows construction of glucose blood vessels or subcutaneously mandate that such sensing
biosensors without mediators. In addition, enhanced elec- devices have an outside diameter of less than 0.6 mm. Both
tron transfer between proteins and carbon nanotubes results electrochemical and optical sensor technologies have been
in lower overpotentials and higher peak currents observed used to create devices of the required size. These are basically
for the voltammetric response of several molecules at elec- miniaturized versions of the previously described in vitro
trodes modified with carbon nanotubes.120 For DNA sensors, electrochemical and optical sensor devices. However, in addi-
using carbon nanotubes in a detector for the hybridization tion to their small size, such devices must exhibit stable out-
event provides an efficient way to amplify the label-free elec- put signals because reliable calibration of the probes with
trochemical detection of DNA hybridization and to enhance calibrating solutions is not possible once the probes are
charge transfer between surface-anchored DNA sequences inserted. Although so-called in situ calibration is possible
and carbon nanotubes.91,122 (periodically removing an in vitro blood sample to obtain
Nanomaterial labels, including Au, Ag, and semiconductor current values of analytes and updating in vivo sensor output
nanoparticles, have been shown to result in large signal en- to this value), if the frequency of such in situ calibrations is
hancements and to lower limits of detection in electrochemi- high, the true advantage of having an in vivo probe is greatly
cal immunosensors for disease-related protein biomarkers, diminished.
and for detection of DNA hybridization events.123 Au Continuous in vivo measurement of oxygen saturation
nanoparticles (AuNP) have been used as both immobiliza- (SaO2) has been achieved by placing small optical fibers into
tion platforms and labels for electrochemical immunosen- the bloodstream via a catheter and then measuring the reflec-
sors.124 Electrodes with layers of densely packed 5-nm AuNP tive absorbance of the blood at two or three appropriately
were used as a matrix for immobilization of horseradish per- selected wavelengths based on the absorbance spectra of oxy-
oxidase in a sandwich immunoassay for PSA with a detection and deoxy-Hb.133 In addition, implantable analytical sensors
limit of 0.5 pg/mL.125 In an optical assay, oligonucleotide that provide continuous readings of blood gases (pH/PCO2/
targets labeled with AuNP instead of fluorophore probes have PO2), when inserted within the radial artery, especially for
been shown to enhance sensitivity toward the oligonucle- critically ill patients already fitted with an arterial line, have
otide target by up to two orders of magnitude.126 A commer- been developed.134 Such sensors are usually based on the clas-
cial system using AuNP for electrochemical DNA sensing is sical Clark-style design, in which O2 is reduced at a micro-Pt,
the Verigene System, which was reviewed previously. -Ag, or -Au working electrode confined (along with Ag/AgCl
Other examples of nanotechnology applied to chemical reference) within a narrow-diameter gas-permeable catheter
sensing are emerging. For example, nanopores down to 1.4 tube made of a given polymer (Fig.17.17A), with the result-
nm in diameter have been created in lipid bilayers through ing current being proportional to the PO2 in the medium
the action of the protein a-hemolysin. A pore with nanome- surrounding the catheter. Indwelling electrochemical pH and
ter-scale dimensions has a size comparable to that of a single PCO2 sensors are typically potentiometric devices, based on
large molecule (protein, single-stranded DNA). The mole- polymer membrane pH electrode technology or the use of
cule must be charged to be driven through the pore by an solid-state metal oxide–based pH sensors. Incorporation of
electric field. The contribution of the large molecule to the lipophilic proton ionophores (e.g., tridodecylamine; see
passage of electric (ionic) current through the pore is small Fig.17.3) within the walls of plastic tubing provides a conve-
compared with that of mobile small ions, allowing detection nient means of preparing a novel dual-lumen pH/PCO2 sens-
of the target analyte down to a single molecule.127 Microfab- ing design that has been demonstrated in animal experiments
ricated cantilevers made of silicon or silicon nitride have to provide accurate in vivo results.135
been used as transducers in affinity biosensors for monitoring However, miniaturization of electrode designs that enable
(1) DNA hybridization, (2) antigen–antibody interactions, several sensors to be bundled into a single implantable device
or (3) absorption of bacteria (see Chapter 27). Mechanical remains a significant engineering challenge. Consequently,
bending of the cantilever on the order of nanometers, in many efforts aimed at developing commercially viable intra-
response to affinity reactions, is monitored. Although the sen- vascular blood gas sensors for simultaneously monitoring
sitivity of cantilever biosensors is limited by nonspecific bind- pH, PCO2, and PO2 have used modern optical fiber–based
ing, the technology is equivalent in sensitivity to that of other technology alone or in combination with a single electro-
label-free methods, for example, in the picomolar range for chemical device. Because the outer diameter of available fi-
detection of oligonucleotides, and in the nanogram per millili- bers continues to decrease, it is now possible to bundle three
ter range for measurement of antigens.128 or more separate chemically sensitive fibers within a single

Ag/AgCl Internal electrolyte

electrode solution

Silicone, polyurethane, or Platinum or

A polyethylene tubing silver cathode

Sensing windows positioned

radially around fiber to
Thermocouple pH sensing fiber minimize wall effect

Outer casing composed PO2 PCO2 sensing fiber encased

of microporous polyethylene electrochemical in bicarbonate solution
membrane with covalently sensor
B attached heparin

To recording device Inserted into tissue

Ag/AgCl reference electrode coil Sensing membrane PTFE

with glucose oxidase insulation
C over Pt-Ir electrode
FIGURE 17.17 Schematics of various implantable electrochemical and/or optical sensors useful for
continuous in vivo monitoring. A, Catheter-style amperometric oxygen sensor. B, Design of Paratrend
intravascular combined partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), and
pH sensor (hybrid electrochemical/optical design). C, Needle-type electrochemical glucose sensor useful
for monitoring glucose subcutaneously to track blood glucose concentrations continuously. Ag/AgCl,
Silver-silver chloride; Pt-Ir, platinum-iridium; PTFE, Teflon.

catheter with an outer diameter of less than 600 mm for Despite significant efforts by a number of large biomedi-
implantation within human radial arteries, without damp- cal companies in the 1980s and 1990s, only the Paratrend
ening the pressure waveform detected by a microelectronic probe was commercially available for a period of time for the
pressure transducer within the arterial line. Absorbance-, intravascular measurement of blood gases. As shown in
fluorescence-, and phosphorescence-based chemistries have Fig.17.17B, this indwelling sensor consisted of a novel hybrid
been investigated in the design of sensors with suitable se- design, in which O2 was sensed electrochemically (catheter
lectivity and calibration stability.132 Appropriate indicators form of Clark sensor), whereas pH and PCO2 were deter-
are usually immobilized on the distal ends of the fibers, mined via fiber-based optical fluorescence sensors.136
although alternate configurations and/or locations have Although acceptable clinical performance of the Paratrend
also been proposed. Most optical O2 measurements are was reported, frequent in situ recalibrations were suggested.137
made with indicators whose luminescence is quenched in A later version of this product replaced the electrochemical
the presence of O2, whereas pH sensors are prepared by O2 sensor with an optical fiber design. However, continued
immobilizing pH indicators (e.g., phenol red) in hydrogel- problems with in vivo performance and concomitant costs of
type films.53 Optical sensors for PCO2 can be easily pre- using the device prevented widespread use, and the product
pared from the same pH sensors by incorporating bicar- is no longer available.
bonate salt in the hydrogel layer and then covering this Implantable glucose (and lactate) sensors have been based,
layer with a gas-permeable polymeric film (usually silicone almost exclusively, on electrochemical transducers. For
rubber material), as described previously in the section on example, one such sensor is based on using dual O2 sensors
Optical Sensors. in a single catheter design for indwelling blood glucose and

lactate measurements.138 One O2 sensor with immobilized Biocompatible polymeric coatings are traditionally employed
glucose or lactate oxidase provides response to substrate con- in addressing the biofouling issue.150 Additionally, polymeric
centrations (decreasing surface O2 in response to increased coatings with embedded anti-inflammatory drugs such as
glucose or lactate), whereas the second matched O2 sensor, Dexamethasone are used to mitigate sensor biofouling dur-
without enzyme, is able to correct for unknown and varying ing use.151 One new approach in this direction uses novel NO
amounts of endogenous PO2. Others have focused on the release and/or generating polymers to coat the surface of in-
design of probes with amperometric detection of H2O2 (via travascular sensors.152–154 The potent antiplatelet activity of
oxidation) at an underlying iridium/Pt anode, similar in op- NO has been shown to greatly reduce the formation of
eration to those used in vitro in commercial instruments.139 thrombus on the surface of implantable electrochemical O2
With such designs, the use of outer polymer films to restrict sensing catheters and to yield much more accurate continu-
glucose (or lactate) diffusion relative to O2 is critical to ous PO2 values in animal experiments. Furthermore, NO has
achieve a linear electrochemical response to glucose from been shown to decrease the inflammatory response that oc-
normal (90 mg/dL; 5 mmol/L) to increased concentrations curs for glucose sensors implanted subcutaneously155,156 and
found in patients with diabetes (greater than 500 mg/dL; for glucose and lactate sensors implanted intravenously.157
27.8 mmol/L). Fig.17.17C illustrates a design in which the In 2000, the Food and Drug Administration (FDA) ap-
needle-type probe is constructed by multiple membrane proved the first CGM system from MiniMed (currently part
coatings and electrodeposition of the glucose oxidase layer.140 of Medtronic, Northridge, CA) and had a mean absolute
Designs similar to this are among the currently US Food and relative difference (MARD) of around 25% between glucose
Drug Administration–cleared sensors available for subcuta- value measured by YSI glucose and the CGM sensor. Addi-
neous monitoring of glucose.141–143 In one instance, the sen- tionally, MiniMed sensor required periodic calibration using
sor is actually fabricated on a narrow planar substrate, rather an off-line fingerstick glucose from a single-use blood glu-
than on a cylindrical wire-based system. Relatively frequent cose sensor to correlate patient’s glucose to CGM signal. FDA
calibration of all such sensors is still required via periodic in approved new devices from Abbott (Chicago, IL) in 2017 and
vitro blood tests, especially in the early stages after subcuta- from Dexcom (San Diego, CA) in 2018 offer calibration-free
neous implantation.141 or factory-calibrated sensors with MARD of 11.7% and 9.0%,
respectively. Abbott, Freestyle, CGM consists of a sensor
CONTINUOUS GLUCOSE MONITORING (CGM) patch that is worn on the upper arm and measures glucose in
ISF every 15 minutes and has 14-day use-life. When users
SENSORS scan the monitor over the sensor patch, it reports current
Single use electrochemical sensors test strips with capillary reading and 8-hour trending of glucose. The Dexcom, G6
blood testing has been the routine methodology for frequent CGM measures glucose every 5 minutes, and the results are
self-monitoring of blood glucose (SMBG) at home by diabetic wirelessly communicated via Bluetooth technology to a mon-
patients. Recently, a new class of glucose sensors designed to itor, cell phone, or an electronic watch, and it has a use-life of
help manage patients with diabetes by continuous monitoring 10 days. Newer versions of Medtronic CGM, the Guardian
of glucose in interstitial fluid (ISF) were introduced to the connect, has 7-day use-life and requires calibration with fin-
market.144,145 Although SMBG has been the standard of glu- ger sticks every 12 hours. All three devices are based on the
cose testing at home, CGM is rapidly increasing in recent years same electrochemical glucose biosensor principles described
due to the advances in wireless technologies, miniaturization, in the biosensors section of this chapter. One drawback of
development of calibration-free sensing technologies, and these ISF-based CGM sensors is that they are (worn either on
push for reducing finger pricks for self-monitoring.146,147 upper arm or on the abdomen) are susceptible to erroneous
Continuous monitoring of glucose in ISF first appeared glucose results from the prolonged pressure on the sensor
commercially in the late 1990s. Currently, devices from multi- during sleep.158 Pressure on the sensor can induce changes at
ple manufacturers are offered on the market. Subcutaneously the interstitial space due to decreased tissue perfusion, oxy-
implanted glucose sensors, in the earlier designs, required pe- gen tension, or change in temperature altering glucose re-
riodic calibration using results from fingerstick blood glucose sponse of the CGM sensor. Additionally, an adhesive patch is
and required replacement every 3 to 7 days, depending on the used for holding the sensor on the upper arm or on the abdo-
device. Despite limited commercial success in the early phase, men and the adhesive patch can peel off from the skin caus-
continuous glucose monitors, based on subcutaneously im- ing less than expected use-life. Another new CGM is the
planted sensors, have been shown to detect and predict hypo- Eversense (Senseonics, Germantown, MD) system approved
glycemic events and reduce time spent in the hypoglycemic by the FDA in 2019; it is an optical sensor that requires hos-
range in adults and children with type 1 diabetes.148 Several pital installation under the skin of the upper arm. This de-
clinical trials have shown significant reduction in glycosylated vice measures glucose every 5 minutes and has use-life of
hemoglobin concentration among users of continuous glucose 90 days.159,160 This new Eversense system provides the longest
monitors.148,149 CGM with a MARD of less than 8.5% and eliminates the er-
Sensor technologies used in these ISF implanted devices rors from localized pressures and patch adhesive issues. Ever-
are similar to glucose oxidase-based biosensors described in sense CGM is based on a fluorescence transduction method
the biosensors section above with additional process steps to and uses a nonenzymatic boronic acid–glucose binding poly-
address biofouling during the use. To improve biocompati- mer as the base principle for measuring glucose. Binding of
bility of these sensors when implanted in ISF, significant ef- glucose increases fluorescence signal proportionally to the
forts were needed to develop coatings to reduce the biological concentration. However, the Eversense sensor assembly is
response to yield continuous analytical results that closely bigger in size (millimeters) than the typical electrochemical
match those attained by conventional in vitro test methods. sensors that are micron sized.

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M U LT I P L E CHOICE QUESTIONS 6. Which of the following is the fundamental quantity mea-

sured by an ion selective electrode?
1. Which of the following is considered a source of error for a. Fugacity
hematocrit measurements by conductivity? b. Concentration
a. High glucose concentration c. Water content
b. Low PO2 concentration d. Activity
c. Abnormal protein concentration e. Current
d. High hemoglobin concentration 7. Which of the following is a possible interfering substance
e. There are no known sources of error for this measure- for a Clark-style amperometric PO2 sensor?
ment a. Sodium because it can change the conductivity of
2. Blood K1 concentration is routinely measured using the cell
which of the following electrochemical techniques? b. PCO2 because it is an acidic gas
a. Potentiometry c. Glucose because it consumes oxygen in the presence
b. Amperometry of glucose oxidase
c. Conductometry d. High protein concentration which may foul the plati-
d. Coulometry num electrode
e. Polarography e. An electroactive anesthesia gas diffusing across the
3. Which of the following may be used to correct the output gas permeable membrane
of a potentiometric sensor for contributions from inter- 8. Which of the following analytes are measured by enzyme
fering ions? electrode-based biosensors?
a. Nernst Equation a. Glucose, lactate, and Creatinine
b. Nikolsky-Eisenman Equation b. Sodium
c. Faraday’s Law c. Potassium
d. Stern-Volmer Equation d. pH
e. Debye-Huckel Equation e. PCO2
4. Affinity biosensors are usually single-use devices for what 9. What is the primary analyte recognition component in
reason? immuno-sensors?
a. The sensors are produced inexpensively using modern a. Ionophore
fabrication techniques b. Diffusion membrane
b. High binding constant limits reversibility of the affin- c. Glucose
ity reaction d. Antibody
c. Enzyme activity is destroyed during the binding reaction e. Electrode
d. Transducer material is irreversibly fouled by blood 10. Continuous glucose monitoring devices measure glucose
proteins in which of following fluids?
e. Affinity reaction removes recognition element from a. Plasma
sensor surface b. Serum
5. Which of the following may be considered an interfering c. Interstitial fluid
species for measurement of chloride using an anion- d. Arterial blood
exchanger type ion selective electrode? e. Pleural fluid
a. Sodium
b. Albumin
c. pH
d. Salicylate
e. Fluoride

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Electrochemistry and Chemical Sensors: Prasad V.A. Pamidi

Uploaded by

Electrochemistry and Chemical Sensors: Prasad V.A. Pamidi

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17

Electrochemistry and Chemical Sensors*

*The full version of this chapter is available electronically on ExpertConsult.com.

Millivolts vs. Ag/AgCl

ETH 227: Na

ETH 1117: Mg2

Glass electrode shaft

Reference electrode (Ag/AgCl) Internal electrode (Ag/AgCl)

Sodium bicarbonate Phosphate buffer

Sample inlet Sample outlet

pH-sensitive glass membrane

dissociates, shifting the pH of the bicarbonate solution in pH CO2

Sample Solution this case); F 5 Faraday’s constant (96,487 coulombs/mol);

Ag/AgCl cylindrical electrode

N 5 the amount of substance reduced or oxidized in moles; Optical Isolator

MOx GOOx Product

Ag/AgCl MRed GORed Glucose

glucose oxidase to a metallic, amperometric electrode using mV

Microporous nylon Affinity Sensors for DNA Analysis Using

Capture probe Sample Oxidation of guanine on target DNA

available on nanostructures for immobilization of labels and

Ag/AgCl Internal electrolyte

Silicone, polyurethane, or Platinum or

Sensing windows positioned

Outer casing composed PO2 PCO2 sensing fiber encased

To recording device Inserted into tissue

Ag/AgCl reference electrode coil Sensing membrane PTFE

Instead of implanting glucose sensors intravascularly or SELECTED REFERENCES

REFERENCES preparations using potentiometric polyanion sensors. Anal

M U LT I P L E CHOICE QUESTIONS 6. Which of the following is the fundamental quantity mea-

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ETH 227: Na

ETH 1117: Mg2