Anal. Chem.

2004, 76, 3285-3298

Electrochemical Sensors
Eric Bakker

Department of Chemistry, Auburn University, Auburn, Alabama 36849

Review Contents sensors, even though they are classified as microscopic tech-
Potentiometric Sensors 3285 niques. Hyphenated systems, such as microdialysis probes
Reviews 3285 coupled to an electrochemical detection system, optionally after
Theory and Characterization 3286 an on-line separation step, act as sensors as well. The reader of
Detection Limit 3286 this fundamental review must therefore keep in mind that
Component Design 3286 integration between fields is at an advanced stage and many
Membrane Materials 3287 exciting developments cannot be discussed here because of space
Reference Electrodes 3287 limitations and overlap to other topics discussed separately.
Voltammetric Sensors 3287
A 200 reference limit was used, which means that only a
Reviews 3287
fraction of relevant works are covered here. This review attempts
Interrogation Principles 3288
Sensors Based on Electrochemically Driven 3288
to present a relatively good coverage of review articles and a
Extraction selection of original research articles that emphasize new chemical
Electrode and Coating Materials 3289 developments or principles as opposed to solving analytical
Microelectrodes 3290 problems. The ACS SciFinder and, to a lesser extent, the ISI
Electrochemical Gas Sensors 3291 Science Citation Index were used to compile the selection of papers
Reviews 3291 presented here. Subject and author searches were performed, and
Original Papers 3291
for a number of key journals, the table of contents listing was
Biosensors 3292
manually read. Only journal articles written in English were
Reviews 3292
considered. The patent literature and conference proceedings or
Enzyme Biosensors: Glucose 3292
Other Enzyme Biosensors 3293 abstracts were ignored. Electrochemical sensors comprise the
Immunosensors 3294 largest group of chemical sensors. Because of the comparatively
Oligonucleotides: Direct Detection 3294 large number of review articles that have been published and the
Oligonucleotides: Intercalator Detection 3294 breadth of this research topic, this review will not be able to give
Oligonucleotides: Enzyme Amplified 3295 due credit to all the excellent work that is being done in this field.
Oligonucleotides: Nanoparticles and Quantum 3295 This author therefore apologizes to anybody who feels that some
Dots
key papers have been left out.
Conclusions 3296
Literature Cited 3296
POTENTIOMETRIC SENSORS
Reviews. The group of Bachas published a broad review on
This review on electrochemical sensors covers the full calendar
ionophore-based potentiometric and optical sensors, with 149
years of 2002 and 2003. This review is concerned with the status
references, aimed at a more general analytical readership,
of the actual development of electrochemical sensing principles
emphasizing mechanistic principles, recognition elements, and
and covers potentiometric sensors, reference electrodes, voltam-
most important applications (1). Umezawa et al. wrote two
metric sensors, electrochemical biosensors (enzyme electrodes
comprehensive updates of their reference work on selectivity
and affinity-based sensing principles), and electrochemical gas coefficients of ion-selective electrodes (ISEs) that cover papers
sensors. Note that some gas sensing principles are also covered from 1988 to 1998 (2, 3). Besides actual numerical selectivity
in the topic of voltammetric sensors. coefficients, the reviews also report on the methodology of
The area of electrochemical sensors continues to broaden and determination, response slopes, ionophore structures, and chemi-
blend with many other topics, including some for which other cal compositions. The review on inorganic anions comprises 72
fundamental reviews are being written. For instance, electro- pages (references are listed separately on each page) (2), while
chemical principles for the detection of analytes are highly relevant the one on organic ions contains 105 pages (3). Macca wrote a
in microfluidics and the broader field of separation science for critical review on the inconsistencies of published selectivity
the purpose of injection, pumping, valving, and detection. The determinations performed in 2000 and 2001, with 68 citations (4).
moving of droplets by the electrowetting effect is based on He suggested that much of the literature data is still of limited
electrochemical principles. Contactless conductivity detectors are significance to other researchers, despite clearer guidance given
electrochemical detectors, even though they are not sensors in in the past few years. An Analytical Chemistry A-page article was
the classical sense because they lack selectivity. Scanning written on the principles and possibilities of low detection limit
electrochemical microscopy and chemically selective scanning potentiometric sensors (30 refs) (5). Bobacka et al. reviewed the
tunneling microscopy are really spatially resolved electrochemical application of conducting polymers to potentiometric sensors, with
10.1021/ac049580z CCC: $27.50 © 2004 American Chemical Society Analytical Chemistry, Vol. 76, No. 12, June 15, 2004 3285
Published on Web 05/01/2004
230 references (6). Such polymers are primarily used as inner
reference elements, but have been explored as ion-selective
membrane materials as well.
Theory and Characterization. A detailed extension of earlier
work on the theory of so-called non-Nernstian equilibrium
responses of ionophore-based ISEs was presented by Amemiya
et al. (7) Non-Nernstian slopes based on zero current sample
concentration polarizations, which are important for low detection
limit ISEs, were not discussed here. As long as the ionophore
complexes primary and secondary ions independently, the model
shows how the observed electrode slope must change as a
function of the charges of ions and ionophore involved. This
elegant work has important implications for future ionophore and
membrane design.
The determination of stability constants of ion-ionophore
complexes directly in ion-selective membranes has been a chal-
lenge for many years. Among the methods recently introduced,
the so-called sandwich membrane method, which utilizes a
concentration polarized membrane made up of two segments of
known composition, is one of the most powerful. Mikhelson et
al. have made their early work, published in Russian, more
accessible and discussed the method in depth, focusing on their
titration technique, the determination of complex stoichiometries,
and estimation of possible biases such as ion pair formation and
diffusion potential (8). Among other papers, this method was
utilized by Ceresa et al. to characterize the binding properties of
a highly halide selective ionophore, [9]mercuracarborand-3 (9),
which had been introduced earlier by Bachas and Hawthorne.
The complex formation constants observed for this ionophore
corresponded quantitatively to extraction constants determined
with optical sensors based on the same ionophore, showing once
more that diffusion potentials are generally not significant with
ion-selective membranes. The protocols for determining complex
formation constants by this method were extended to the
quantification of membrane acid dissociation constants of H+-
selective ionophores and formation constants for electrically
charged anion ionophores (10). The sandwich method was also
found to be useful for the quantification of electrolyte coextraction
processes (11). When one membrane segment contained a cation
exchanger and the other an anion exchanger, the observed
potential of the combined sandwich could be related to the
coextraction constant of the respective electrolyte, which in turn
was used to successfully predict the upper detection limit of
ionophore-based ISEs.
While the sandwich membrane method mentioned above has
been used earlier for the determination of ionic impurities in ISE
membranes, Gyurcsanyi and Lindner have used an optical method
to do the same, where the level of protonation of a dilute
electrically neutral lipophilic pH indicator in the membrane is
quantified (12). This method works when ion-exchange and
electrolyte coextraction processes are experimentally excluded.
De Marco and co-workers continued to apply an array of
surface characterization techniques to the understanding of ion-
selective electrodes. In one example, the fouling of mercury(II)
chalcogenide electrodes upon prolonged contact in saline solution
was studied by XPS, SIMS, RDE-EIS, and SR-GIXRD, and it was
found that fouling is linked to the poisoning by silver salts (13).
3286 Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
Detection Limit. The discovery that the detection limit of
ionophore-based ISEs can be lowered to trace levels has initiated
interesting research by a number of groups. A steady-state model
was developed to predict the lower detection limit of ionophore-
based ISEs (14). Importantly, this model allows the experimental-
ist to calculate optimal inner solution compositions so that rational
design becomes possible. Experiments with silver-selective mem-
branes correlated well with predictions when an ion-exchange
resin was used to buffer the inner solution, to avoid undesired/
unknown extraction processes from the inner membrane side. The
detection limit of anion-selective electrodes was also evaluated
(15). It was found that the inner solution plays a minor role with
ionophore-free membranes and that the detection limit of an
iodide-selective electrodes based on the [9]mercuracarborand-9
ionophore could be lowered to nanomolar levels. In all cases,
hydroxide was found to be the primary sample interference that
dictates the detection limit via counterdiffusion fluxes. Michalska
et al. have published papers on lowering the detection limits of
ISE with a conducting polymer solid contact (16). The earlier
water layer theory of Pretsch was not considered here. Rather, it
was argued that the self-discharge of the conducting polymer
caused the very high detection limit ordinarily observed with such
systems and that an anodic coulometric control could be used to
reduce the detection limit by ∼3 orders of magnitude to submi-
cromolar levels. The observed detection limits are still much
higher than that observed with optimized liquid contacts, however.
The group of Pretsch found that the use of microspheres normally
used in HPLC, placed on the sample side of ISE membranes,
completely eliminated undesired super-Nernstian response slopes
to give subnanomolar detection limits for calcium (17). Pretsch
also used a rotating electrode configuration, with the membrane
placed off-center of the rotating axis, to yield detection limits in
the picomolar range and rapid response times (18). In other work,
an electrode rotator was used as a diagnostic tool to evaluate the
level of optimization of the ISE in terms of its detection limit (19).
De Marco’s group studied the effect of diffusion fluxes on the
detection limit of the commercially available jalpaite copper ion-
selective electrode (20). Indeed, rotating disk electrode experi-
ments revealed that the detection limit could be lowered to about
nanomolar levels. The detection limit could be further decreased
by using modified membrane compositions to further minimize
copper dissolution, using an excess of sodium sulfide.
Component Design. Polymer membrane-based ISEs require
ion-exchanger properties, and historically, tetraphenylborate de-
rivatives have been incorporated for cation-selective electrodes.
Unfortunately, they have limited chemical stability and lipophilicity
and are very difficult to covalently anchor onto the polymeric
backbone. In response to this problem, perhalogenated closo-
dodecacarboranes have been introduced as alternative compounds
in ISE membranes, with characteristics that equal or surpass that
of the best available tetraphenylborates (21). In subsequent work,
this class of compounds was covalently attached onto a meth-
acrylic copolymer, and the ion selectivity of the membrane was
perfectly retained (22). On the basis of this ion exchanger, a
calcium-selective membrane based on a plasticizer-free polymer,
with chemically attached calcium ionophore and cation exchanger,
was successfully fabricated for the first time (22).
 It is known that calcium-selective ionophores form very stable
complexes in the membrane. For this reason, the resulting
membranes are often subject to electrolyte coextraction at elevated
concentrations, which leads to anion interference. The group of
Nam has introduced a new tweezer-type ionophore with much
lower complex formation constants (23). While the resulting
selectivity is somewhat inferior to that of the best available calcium
ionophores, anion interference was completely eliminated, even
in calcium perchlorate solutions up to 0.1 M. Similarly, a new
polymerizable derivative of ETH 129 was shown to form weaker
complexes than its parent molecule ETH 129 (24). Membranes
containing the covalent attached calcium ionophore showed
somewhat inferior selectivity as well, but a reduced anion interfer-
ence. Importantly, the covalent anchoring of this ionophore
resulted in Nernstian response slopes, even with a calcium-free
inner solution that would otherwise induce a strong inward ion
flux. This was attributed to the reduced calcium mobility in the
membrane (24).
Different molecular design strategies were used by Sasaki et
al. for the recognition of the ammonium ion (25). A tripodal
preorganization to reject the spherical potassium was successful,
but the resulting membrane suffered from calcium interference.
The use of crown ethers containing bulky decalino blocking units
gave the best results, with membranes that have better ammonium
selectivity than any system reported to date.
Uranyl salophenes have been established as very promising
phosphate ionophores but have traditionally suffered from chemi-
cal decomposition in contact with phosphate solutions. Wojciechows-
ki et al. have used a modified inner solution, in analogy to work
on low detection limit ISEs, that effectively buffers both phosphate
and uranyl ions (26). The resulting ion flux in the direction of
the inner solution keeps the membrane mostly phosphate-free,
thereby increasing the lifetime of such membranes to over two
months.
Bobacka and co-workers explored the recognition of aromatic
cations such as N-methylpyridinium based on π-coordinating
carriers that were either electrically charged or neutral, in
membranes containing different plasticizers and backside con-
tacted with a polythiophene-type solid contact (27). Only electri-
cally charged carriers were found to show significant changes in
selectivity.
The group of Nam has assessed so-called tweezer-type carbon-
ate ionophores to the measurement of carbon dioxide in seawater
(28). With one of the ionophores, a very high carbonate selectivity
over chloride (log Kpot, -6) and other minor ions was observed,
sufficient for direct determination in seawater without sample
pretreatment. A comparison of seawater analyses to reference
methods was successfully performed.
Malinowska et al. studied zirconium(IV)porphyrins as electri-
cally charged ionophores for fluoride (29), which was preferred
over all other tested ions, including the lipophilic perchlorate. A
super-Nernstian response slope was observed because of hydroxy-
bridged porphyrin dimer formation as confirmed by UV/visible
spectroscopy.
Membrane Materials. Kimura et al. have continued to study
their thermotropic liquid crystals as ion-selective membranes
doped with crown ether ionophores (30). They found that an
ordered arrangement of the ionophore (confirmed by polarized
IR spectroscopy and X-ray diffraction) went along with significantly
enhanced ion selectivity, which they explained with cooperative
interaction of adjacent crown ethers.
Polymeric membranes modified with Zeolite particles were
used by the group of Walcarius for the preparation of ammonium-
selective ISFETs (31). While a very high Zeolite content was
needed (43 wt %) and the observed electrodes slopes were
severely sub-Nernstian, a low detection limit of 10 nM was
reported.
An anodically electrodeposited iridium oxide pH microelec-
trode was fabricated and characterized by Bezbaruah and Zhang
(32). While response times were fast and the pH measuring range
was large, interference from redox-active compounds was signifi-
cant. Yamamoto et al. fabricated a tungsten nanoelectrode (with
400-800-nm tip diameters) by etching a tungsten wire followed
by electrooxidation to tungsten oxide (33). The electrode, which
gave a measuring range between pH 2 and 12, was applied to
extracellular pH measurements on endothelial cells.
The group of Bachas used an ISE membrane covered with a
polymer containing phosphorylcholine functionalities, which mimic
the polar groups on cell surfaces (34). Decreased adhesion and
activation of platelets was demonstrated by immunostaining, and
ISE sensing characteristics were not affected by the coating. See
also Voltammetric Sensors for other approaches to biocompat-
ibility.
A novel potentiometric measuring principle for the detection
of saccharides was developed on the basis of poly(aniline boronic
acid) (35). The complexation resulted in a change of the effective
pKa of the polymer, which gave rise to a change in the observed
potential. Additives such as Nafion or sodium fluoride also had a
marked effect on sensor sensitivity.
van der Wal et al. introduced a technique for the simple
covalent attachment of poly(vinyl chloride) membranes to solid
substrates such as glass and other oxide surfaces (36). Mem-
branes attached via a silane coupling agent containing an amine
group and a heating step showed excellent adhesion properties
and retained their sensing characteristics relative to membranes
cast without linking agent. This work potentially solves a long-
standing problem in the development of ion-selective field effect
transistors, without the need for a new polymeric material.
REFERENCE ELECTRODES
Very few works reported on new reference electrode principles.
Langmaier and Samec explored freshly polished copper wires
directly inserted into an electrolyte solution of the solvent
o-nitrophenyl octyl ether as a junction-free inner reference
electrode in ion-transfer voltammetry (see topic below) (37). They
reported impressive stabilities on the order of 2 mV over 300 h,
but the exact mechanism of this behavior could not be explained.
Lee and Sohn explored field effect transistor-type reference
electrodes consisting of a one ISFET gate covered with a pH-
insensitive polymer double layer that was used in conjunction with
a pH ISFET for pH detection (38). The mechanism for its
functioning remains unclear as well.
VOLTAMMETRIC SENSORS
Reviews. Wang reviewed the current status and future
challenges of miniaturizing electroanalytical systems, their incor-
poration into microfluidic devices, and their application to point
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004 3287
of care and environmental analysis as well as genetic testing, with
17 references (39). In another review, the same author stressed
the usefulness of electrochemical sensors for environmental
monitoring applications as an approach to a greener analytical
chemistry compared to traditional instruments (35 refs) (40). The
group of Wightman wrote an A-page article on their work on the
development of electrochemical sensors for the neurotransmitter
dopamine, with 30 citations (41). Within a special issue of TrAC,
Trends in Analytical Chemistry on microelectrodes and micro-
dialysis probes for neuroanalysis, Wightman’s group also dis-
cussed characterization and validation procedures and required
selectivities of such microelectrodes when used for the measure-
ment in the brain (22 refs) (42). The status of development of
electrochemical arsenic sensors for environmental monitoring
applications was reviewed by Feeney and Kounaves as part of a
special Talanta issue on arsenic detection, including their own
work on portable systems based on microfabricated gold arrays
(35 refs) (43). The status of electrochemical sensors for oc-
cupational and environmental health applications was reviewed
by Ashley with 109 citations, with an emphasis on rugged and
miniature electroanalytical devices for on-site monitoring (44).
Methods such as disposable screen printing technology for the
fabrication of sensors for trace metal pollutants in a variety of
sample matrixes was reviewed by Honeychurch and Hart (46
citations) (45). The status of electrochemical sensors for the
detection of metal pollutants in coastal waters was reviewed by
Achterberg and co-workers, with 17 references (46). The history
and current status of electrochemical nitric oxide sensors based
on modified electrodes was reviewed by Bedioui and Villeneuve
(129 citations) (47) as well as Ciszweski and Milczarek (with 32
refs, in a special NO detection issue of Talanta) (48). The
application of self-assembled monolayers as a bottom-up fabrica-
tion principle for the realization of electrochemical sensors for
pH and inorganic and biological sensors has been reviewed by
Gooding et al., with 168 citations (49). This paper includes a
discussion of emerging trends such as nanotubes, dendrimers,
and nanoparticles for electroanalysis. Similarly, Hernandez-Santos
et al. reviewed the use of metal and semiconductor nanoparticles
for use in electroanalysis (59 citations) (50), Li et al. reviewed
the electrochemistry at carbon nanotube electrodes in view of
sensor and assay development (51), and Sherigara et al. reviewed
electrocatalytic properties of nanotubes and fullerenes in view of
developing electrochemical sensors, with 230 citations (52). Swain
reviewed the status and future prospects of diamond science for
numerous emerging technologies including electrochemistry and
electroanalysis (53). The status of the development of electro-
chemical sensors based on molecularly imprinted polymers was
reviewed by Piletsky and Turner (45 citations) (54). Similarly,
Merkoci and Alegret reviewed the use of molecularly imprinted
polymers in capacitive, conductometric, voltammetric, and poten-
tiometric sensors (28 refs) (55). The group of Guilbault sum-
marized the status of chemometrics for electrochemical sensors,
with 78 citations, by focusing on multivariate calibration, clas-
sification, pattern recognition, and signal processing (56). Hei-
neman’s group reviewed their own work on spectroelectrochem-
ical sensing, where electrochemistry, spectroscopy, and selective
partitioning are combined into a single sensing device for
improved selectivity (43 refs) (57). The group of Martin reviewed
3288 Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
their work on the development membranes containing pores of
molecular dimensions for new separation and electrochemical
sensing applications, with 45 citations (58).
Interrogation Principles. The groups of Bachas and Grimes
introduced a novel measuring principle to monitor electrochemical
processes without the need for electrical connections (59). Here,
magnetoelastic alloy films were used as the working electrode in
an electrochemical cell and the mass change on the electrode
(polypyrrole deposition) was monitored via magnetic monitoring
of the resonance frequency.
Heineman continued his work on spectroelectrochemical
sensing (see also review cited above (57)), for example, by
describing a sensor for the detection of iron(II) (60). Here, a
Nafion film loaded with a bipyridine ligand was used to extract
iron(II) and to render it strongly absorbing by complexation.
Electrochemical oxidation of this complex again rendered it
colorless. In another example, the stripping voltammetric detection
of lead and cadmium was monitored spectroscopically at the same
time, with separate wavelength regions used for each of the two
metals (750 nm for lead and 400 nm for cadmium), giving added
selectivity (61).
Ekeroth used interfacial capacitance measurements to monitor
the interaction of phosphate monolayers with calcium and
magnesium ions (62). Monitoring a non-Faradaic process, as done
here, appears to be more susceptible to effects unrelated to the
desired molecular interaction and should be used with caution.
Indeed, this approach is an alternative to others who have used
voltammetric techniques with a redox marker for quantifying
surface recognition events. For example, Choi et al. used a
competitive adsorption of electrochemically inactive organic
molecules such as glucose with ferrocene onto a self-assembled
monolayer containing a thiolated cyclodextrin (63). The oxidative
current for ferrocene was indeed reduced with higher sample
concentrations of glucose. A critical selectivity study of this device
was not performed, however.
Baca et al. coupled anodic stripping voltammetry online to
ICPMS to develop a hyphenated technique with high selectivity
and sensitivity (64). It was found that the electrochemical
preconcentration gave detection limits down to sub-ppt levels,
lower than possible with conventional ICPMS, and that it could
be used to eliminate matrix effects as well.
The group of Martin explored the use of nanotube membranes
as ligand-gated ion channel mimics (65). Ion current through the
membrane could be switched on or off by adding hydrophobic
ionic species to the sample that could interact with the hydro-
phobic pores of the membrane that would otherwise be insulating.
Sensors Based on Electrochemically Driven Extraction.
The electrochemically controlled extraction of ions into sensing
polymers and other water-immiscible phases is an attractive
approach to chemical sensing that bridges the fields of polymer
membrane-based ion-selective electrodes and voltammetry at
metal electrodes. Wu et al. used electrochemical control of
conductive polypyrrole films to extract, preconcentrate, and desorb
ionic analytes, which were subsequently analyzed by flow injection
analysis and mass spectrometry (66). The method was found to
work for a variety of cations and anions and appears to be versatile.
Janata’s group used cyclic voltammetry to control the exchange
of chloride ions between polypyrrole and the buffer to fabricate a
label-free DNA hybridization detector (67). The probe DNA was
immobilized onto polypyrrole via magnesium bridging complexes,
and the hybridization event caused a change in the voltammetric
behavior of the film.
In other work, a new measurement protocol was introduced
for ion-selective membranes that lack ion-exchanger properties
(68). Here, current and potential pulses were alternated to control
the extraction processes of the membrane electrochemically. The
resulting responses have the same look and feel as potentiometric
membrane electrodes, but the selectivity and response features
can be tuned and even reversed, and the reversible detection of
analytes that ordinarily give irreversible sensing responses
becomes possible. As an important early example of this approach,
the reversible detection of the polycation protamine was demon-
strated for the first time (69). In parallel work, Amemiya and co-
workers used cyclic voltammetry on micropipets to demonstrate
the detection of protamine (70). In a similar effort, Samec et al.
used cyclic voltammetry for the electrochemical detection of the
anticoagulant polyanion heparin (71).
The analogy of ion-transfer voltammetry to potentiometric ion-
selective electrode response was also stressed by Wooster et al.,
who studied microparticles containing 7,7,8,8-tetracyanoquin-
odimethane and tetrathiafulvalene in contact with electrolyte
solutions. The voltammetric waves changed as a function of the
type and concentration of electrolyte and were explained by ion
incorporation processes as well (72). Long and Bakker used
normal pulse voltammetry on pH-sensitive polymer membranes,
and an apparently Nernstian relationship between sample pH and
half wave potential was also observed that correlated closely with
that of corresponding ion-selective electrodes (73). Spectral
imaging experiments confirmed the electrochemical results. This
work forms the precursor for the pulsed galvanostatic approach
mentioned above where the potential can be directly obtained from
the experiment.
The group of Buffle continued their work on permeation liquid
membranes as selective preconcentrators for metal speciation
measurements by optimizing membrane and ion channel geom-
etry (74). In this approach, the membrane is a traditional transport
membrane that works on the basis of zero current counterdiffusion
fluxes for the transport and preconcentration of metal ions at the
backside of the membrane for metal ion sensing. See the topic
above of detection limits of ion-selective electrodes for similar
mechanisms.
Rahman et al. used a hybrid between ion extraction/recogni-
tion and redox electrodes by doping a thiophene-based conducting
polymer, which is normally known for its electrochemically
mediated extraction properties, with EDTA (75). The polymer was
coated onto a glassy carbon electrode, and the metals lead(II),
copper(II), and mercury(II) were deposited and subsequently
reduced at the electrode, with detection limits in the subnanomolar
range.
Electrode and Coating Materials. Ultrasonic cavitation was
used by Cordero-Rando et al. to fabricate a sol-gel graphite-based
electrode material from an acidic aqueous solvent in view of
developing electrochemical sensors (76). The group of Collinson
used an electrodeposition process from a tetramethoxysilane sol
to fabricate sol-gel silicate films that were rougher than spin-
coated films. Various redox molecules were electroencapsulated
during film formation (77). In other work, Khoo and Chen
encapsulated methylene blue into a similar sol-gel film on glassy
carbon electrodes for the electrocatalytic determination of ascorbic
and uric acid (78). The simultaneous determination of these two
analytes in human urine samples was demonstrated. Sol-gel
technology was also used by the group of Mandler to design an
molecularly imprinted polymer for iron(II) using a tris(2,2′-
bipyridine) complex (79). However, the achieved selectivity was
not satisfactory, suggesting that the recognition and detection of
organic molecules is currently a more successful approach with
this technology.
Domenech et al. showed that Zeolite Y containing an encap-
sulated triphenylpyrylium ion exhibits a markedly improved
oxidative response to dopamine while inhibiting the oxidation of
negatively charged interferences such as ascorbate (80). A 100-
fold excess of ascorbate could be tolerated in a differential pulse
detection mode. The group of Walcarius used Zeolites to chemi-
cally modify carbon paste electrodes for improved electroanalytical
properties (81). When Zeolite particles were used instead of the
classical mineral oil binder of carbon paste, or used as an outer
coating, electrodes with improved responses to copper ions were
observed after an ion-exchange accumulation step.
Mesoporous platinum electrodes possess an enlarged surface
area that enhances their catalytic properties for chemical sensing.
Consequently, Evans et al. used such materials for the enhanced
detection of hydrogen peroxide (82), and Park et al. found that
the normally sluggish nonenzymatic glucose response was greatly
enhanced with such electrodes (83). Enhanced electrochemical
sugar detection after HPLC separation was also reported by You
et al. by the use of highly dispersed Ni nanoparticles in a carbon
film electrode, with detection limits that were at least 1 order of
magnitude lower than with traditional Ni electrodes (84). Zen et
al. used copper-plated screen-printed electrodes for the selective
detection of o-diphenols such as catechol and dopamine in the
presence of m- and p-diphenols as well as ascorbic acid under
very mild conditions (-0.05 V vs Ag/AgCl) (85). The enhanced
selectivity was explained by the formation of a cyclic five-
membered complex intermediate at the copper electrode surface.
Copper electrodes were also used by Paixao to determine ethanol
amperometrically in beverages (86). The principle was incorpo-
rated into a flow injection analysis system and used a PTFE
membrane for ethanol extraction followed by oxidation under
alkaline conditions. The comparison of the data from beverage
analyses with gas chromatography gave excellent agreement.
Boron-doped diamond electrodes continue to be adopted for
electroanalysis because of their high stability, low background
current, and wide potential window. Ferro and De Battisti reported
on an unprecedented 5-V potential window in aqueous solutions
using fluorine-terminated boron-doped diamond electrodes (87).
No electroanalytical applications were yet reported with this
material. Rao et al. showed that boron-doped diamond electrodes
are improved detectors for carbamate pesticides after HPLC
separation, offering better electrode stability (88). If a hydrolysis
step was introduced prior to separation and detection, ppb
detection limits were achieved for these analytes. The group of
Swain explored boron-doped diamond films as electrically trans-
parent electrodes on quartz for spectroelectrochemical applica-
tions, obtained by microwave-assisted chemical vapor deposition
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004 3289
(89). The optical and electrochemical properties of the films were
found to be extremely stable, even in harsh environments, and
found to be superior to that of traditional In-doped tin oxide thin
films. In analogy to carbon paste, monocrystalline diamond paste
electrodes were introduced and studied by Stefan and Bairu for
the determination of iron(II) in pharmaceutical preparations (90).
Wang and Musameh incorporated the electrocatalytic advan-
tages of carbon nanotubes into a more rugged configuration by
forming a nanotube/Teflon composite (91). The electrocatalytic
properties of the material toward NADH and hydrogen peroxide
were not impaired, which was used for biosensing of ethanol and
glucose at low potentials by incorporating suitable enzymes into
the electrode matrix. In a similar approach, Valentini et al.
compared carbon nanotube pastes, obtained by oxidative purifica-
tion of such nanotubes followed by mixing with mineral oil, to
traditional carbon paste and found significantly improved elec-
troanalytical properties for the oxidative detection of dopamine
(92).
Wang’s group developed an electrochemical sensor for the
continuous monitoring of the explosive 2,4,6-trinitrotoluene (TNT)
in untreated marine environments with 25 ppb detection limits
(93). The sensor operated by square wave voltammetry at a
carbon-fiber electrode, and oxygen background was corrected for
by a computerized baseline subtraction.
The group of Meyerhoff continued research on nitric oxide-
releasing materials for improved in vivo biocompatibility by
designing an intravascular amperometric oxygen sensor contain-
ing an NO-releasing silicone rubber coating (94). The NO-
releasing diazeniumdiolated secondary amines were covalently
attached to the silicone rubber. In vivo studies of the catheters
over a 16-h period showed no significant platelet adhesion or
thrombus formation, and data from the improved oxygen sensors
correlated well with in vitro values. Robins and Schoenfisch applied
micropatterning techniques to design aminosilane containing sol-
gel surfaces that can release NO to inhibit platelet adhesion while
not interfering with the underlying sensing chemistry (95). The
group of Urban studied the effect of antimicrobial treatments on
the cytotoxicity and cytocompatibility of biosensor membranes
based on polyurethane, with glucose biosensors as a model system
(96). While toxicity of membrane eluates could be eliminated by
washing steps, even after a chemical treatment, the rate of cell
growth on the membranes themselves depended on the type of
treatment used.
Zhang’s group developed a nitric oxide sensor with detection
limits down to 0.3 nM by direct and selective oxidation of nitric
oxide by an array of microelectrodes, which was coated with layers
of the cation exchanger Nafion and a commercial nitric oxide-
selective membrane (97). The sensor discriminated about 1000-
fold against dopamine and 10 000-fold against the typical inter-
ferences ascorbic acid and nitrite.
While molecularly imprinted polymers (MIPs) are potentially
highly attractive materials for chemical sensing, few truly selective
sensors have been developed so far. In most successful electro-
chemical cases, the analyte is directly electrolyzed at an electrode
coated with a MIP, which acts as a selective membrane. An
example for this approach was given by Shoji et al., who developed
an atrazine sensor based on a MIP composed of methacrylic acid
and a cross-linker on a gold electrode (98). While the measuring
3290 Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
range was only in the millimolar range, selectivity was clearly
improved relative to an uncoated gold electrode. See the work of
Rhaman et al. discussed above for other types of selective coatings
(75). Willner’s group used imprinted membranes as coatings on
field-effect transistors for the detection of triazine herbicides (99).
Although sensitivities were rather small, large selectivity changes
and reversals were obtained upon imprinting with various herbi-
cide substrates, making this a promising technique.
Herzog and Arrigan explored various self-assembled mono-
layers, capped with sulfonate and carboxylic groups, on gold
electrodes to reduce surfactant inhibition on the detection of
copper ions by anodic stripping underpotential deposition (100).
While common surfactants had no effect on the calibration curves,
detection limits for copper were only in the micromolar range.
Building on earlier efforts by others, bismuth film electrodes were
used by the group of Smyth as a mercury-free material for the
simultaneous adsorptive stripping analysis of cobalt and nickel
ions, although detection limits were found to be higher than with
mercury thin films (101).
Microelectrodes. Microelectrodes possess numerous advan-
tages that make them highly attractive in chemical sensor research
and scanning electrochemical microscopy. The groups of White
and Amatore developed nanometer-sized (2-150 nm) platinum
electrodes by electrophoretic coating of etched Pt wires with poly-
(acrylic acid) (102). Fundamental electrochemical studies on such
electrodes showed that as few as 7000 molecules can be detected.
In another approach, Abbou et al. fabricated submicrometer-sized
electrodes by melting the tip of Au microwires with an electric
arc followed by insulation with electrophoretic paint, which was
electrochemically removed just at the very tip (103).
The group of Cooper systematically studied the effects of
microelectrode array geometries (center-to-center spacing and
electrode size) on their voltammetric behavior in view of designing
electroanalytical sensors (104). Loosely packed microelectrode
arrays were found to show improved response times in a
ferrocene-mediated enzyme-linked assay configuration.
In an interesting approach, Baranski applied a high-amplitude
and high-frequency alternating voltage onto microelectrodes to
heat the local environment for enhanced electrochemical detection
(105). Apparently, superposition of this heating waveform does
not interfere with normal electroanalytical measurements. Such
hot microelectrodes possess special promise for the detection of
analytes that are kinetically difficult to oxidize or reduce.
Microelectrodes were used in sophisticated arrangements to
probe redox-active analytes in confined samples of biological
relevance. A very important area of research continues to be the
study of neurotransmitters on a single-cell level. The group of
Ewing reported on a liposome model to understand the escape
of transmitters from synapses in vivo, with an emphasis on the
different processes (diffusion vs flow) that dictate transmitter
transport (106). The group of Wightman used a ∼100-pL transpar-
ent fused-silica vial containing a Ag/AgCl reference electrode that
was capped from the outside electrolyte with a drop of oil to study
single-cell uptake processes with carbon fiber microelectrodes that
were inserted into the vial (107). In this elegant work, dopamine
was injected into the vial, which was shown not be depleted by
the continuous fast scan cyclic voltammetry detection unless a
single cell was present that was designed to uptake dopamine.
Cyclic voltammetry was preferred over amperometry to preserve
the analyte in the vial. The same authors showed that cyclic
voltammograms can be deconvoluted to remove the temporal lag
due to adsorption and desorption of catecholamine, leading to
similar effective response times as with amperometry (108). In a
different approach, Yasukawa et al. fabricated a 100-pL cell by
electrochemical back-etching of a sealed gold wire (109). Single
plant cells were then inserted, and cell metabolites released into
the vial were measured with electrochemical enzyme assays. In
a more elaborate approach, picoliter-sized wells approaching the
size of single cells were micromachined onto silicon chips and
the exocytosis of catecholamine was monitored amperometrically
(110). Because of the optimized geometry of the well, a large
fraction of the released catecholamine could be detected with
millisecond time resolution. Extracellular hydrogen peroxide levels
of the brain of living rats were monitored by the group of Michael
with amperometric microelectrodes modified with a cross-linked
redox polymer containing horseradish peroxidase (111). This
work shows that enzyme-modified electrodes can be reliably used,
thereby expanding the range of analytes that can be detected with
such microelectrodes, although this goes at the expense of
temporal resolution. Microelectrodes were also explored by the
group of Compton for the determination of hydrogen sulfide in a
Clark-type configuration where a membrane separates the inner
chamber from the sample (112). The observed current was found
to be independent of the membrane used, which was explained
by the reduced diffusion layer thickness associated with the
microelectrode compared to larger electrode configurations.
MacPerson et al. imaged the diffusion of redox-active probe
molecules through isolated 100-nm-diameter pores of track-etched
membranes by combined scanning electrochemical-atomic force
microscopy with platinum-coated AFM probes (113). This com-
bination of topographical and electrochemical information by a
single probe represents a very attractive tool for spatially resolved
chemical analysis. This paper is just one of numerous examples
dealing with such chemically selective microscopy techniques.
ELECTROCHEMICAL GAS SENSORS
Reviews. Boegner and Doll reviewed the principles of semi-
conductor gas sensors based on the electroadsorptive effect,
where electrical fields applied on the gas-sensitive layer may alter
the adsorption characteristics of the material and hence the
resulting sensing behavior (114). Nicolas-Debarnot and Poncin-
Epaillard wrote a review on polyaniline-based gas sensors, cover-
ing a 7-year period from 1995 (77 citations) (115). Dubbe reviewed
the principles of solid electrolyte gas sensors and their miniatur-
ization to thin-film microsensors (114 citations) (116). Lapham
et al. discussed the difficult task of developing reliable electro-
chemical sensors based on proton conductors for the measure-
ment of dissolved hydrogen gas in molten aluminum (117).
Knauth and Tuller gave a long historical overview of the principles
of solid-state ionics as they relate to a number of important
applications, including gas sensing (292 refs) (118). Ramamoorthy
reviewed the principles and applications of oxygen sensors,
including the solid electrolyte types used for high-temperature
applications as well as dissolved oxygen based on the Clark
electrode and optical sensor principles (72 citations) (119).
Reinhardt et al. reviewed the development of amperometric
sensors for gases other than oxygen, such as NOx, CO, H2, and
hydrocarbons (37 refs) (120). Similarly, Opekar and Stulik
reviewed the status of amperometric solid-state gas sensors with
an emphasis on electrode and electrolyte materials used to achieve
adequate catalytic activity and size of the three-phase boundary
between electrode, electrolyte, and gas (121).
Original Papers. While many gas sensor arrays have been
termed electronic noses in the past few years, research has thus
far focused on the development of gas sensors and the chemo-
metric analysis of the resulting data. The group of Walt has, for
the first time, explored the effect of the nasal cavity flow
environment by constructing a simpler version of such a cavity
as a plastic model (122). While this preliminary study was done
with fiber-optic sensors, it was found that not only the sensitivity
but also the selectivity of the sensor response varies drastically
as a function of position in the nasal cavity.
The group of Zellers recently concluded that even relatively
sophisticated nonspecific gas sensing arrays are not capable of
reliably determining complex, real-world gas mixtures. As a result
of this, sensing arrays capable of distinguishing up to three gases
in a mixture are now developed as a chemically sophisticated
detector in portable gas chromatography devices. In a recent work,
they have characterized chemiresistive vapor sensor arrays on
the basis of spray-coated gold-thiolate monolayer-protected
nanoclusters for the detection of 11 different organic solvent
vapors, with 700 parts per trillion detection limits for most tested
vapors (123).
Dravid’s group used site-specific dip-pen nanopatterning of
precursor inks to fabricate small chemiresistive tin oxide semi-
conductor sensors sensitive to reducing or oxidizing gases (124).
An array of eight different gas sensors was realized with this
technology by doping each ink with different metal ions, giving
different patterns when exposed to single gases such as chloro-
form, toluene, and acetonitrile. As often seen with such nonspecific
sensing arrays, no gas mixtures were tested.
Lewis and co-workers explored the use of plasticizers for their
carbon black-polymer composites for use as vapor-sensitive
detection arrays that are interrogated by resistance measurement
(125). Adding different plasticizer concentrations was found to
alter the selectivity of the polymer as well as the response time,
which may broaden the palette of available materials for gas
sensing. Kaner’s group used polyaniline nanofibers for the
detection of gaseous acids or bases (hydrochloric acid and
ammonia) via changes in the resistance of such fiber assemblies
(126). Such nanofiber films are attractive because of their large
surface area compared to solid film sensors, although it appears
to be difficult to adapt such intrinsically pH-sensitive materials to
a much wider range of gaseous analytes.
Knake and Hauser fabricated an electrochemical sensor for
ozone gas with a 0.6 ppb detection limit (127). The device was
based on a Au-Nafion electrode with a sulfuric acid solution as
internal electrolyte solution. Major interferences such a nitrogen
dioxide were eliminated by use of a chemical filter. The same
group reported on the detection of a mixture of electroactive gases
by using such Au-Nafion electrodes where electrolysis occurs
at a three-phase boundary (128). The accurate analysis of mixtures
of three organic and four inorganic gases was possible in the ppm
concentration range with multivariate calibration and partial least-
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004 3291
squares regression of the results.
The group of de Rooij reported on MOSFET gas sensors with
a modulated operating temperature. When the temperature was
pulsed with a time constant of less than 100 ms, the kinetics of
the gas reactions with the film was found to be modified (129).
This discovery may be used to increase the recovery time after
exposure to a gas such as hydrogen, and temperature cycling may
also be used to discriminate between different gases for multi-
analyte detection purposes.
BIOSENSORS
The field of electrochemical biosensors has seen significant
growth in the past few years, with the development of enzyme
biosensors and DNA detection principles leading the way. The
following papers give just a sampling of the various approaches
that have been explored.
Reviews. An Analytical Chemistry Perspectives article was
published by the group of Turner on the application of natural
receptors in biosensors and bioassays, with 92 references (130).
The authors also outlined the challenges in view of a successful
commercialization of such sensors. Abel and von Woedtke
reviewed the status and challenges of in vivo enzyme-based
glucose sensors (76 citations), emphasizing the importance of the
sensor surface on biocompatibility (131). The group of Heller
reviewed electrochemical sensors based on electrical wiring of
enzymes, including their recent developments of in vivo glucose
sensors as well as immunosensors and DNA sensors (132). Stefan
et al. reviewed the principles of enantioselective sensors by
comparing different electrochemical sensing and recognition
principles (52 citations) (133).
In a special Talanta issue on DNA detection, Palecek reviewed
the electrochemistry of DNA for the detection of DNA damage
and hybridization at attomole levels or lower (120 citations) (134).
Kelly wrote a review on the principles of charge migration trhough
the DNA double helix and their importance to the design of
electrochemical biosensors (135). Fojta reviewed the status of
electrochemical sensors for DNA interactions and damage from
small molecules by use of either intrinsic electrochemical DNA
signals on redox electrodes or electroactive markers that interact
with DNA (158 refs) (136). Similarly, Takenaka reviewed elec-
trochemical techniques based on DNA intercalation by electro-
active probe molecules, including so-called hybridization indicators
(137). Vercoutere and Akeson reviewed the development of
biosensors for DNA sequence detection as a replacement for
established DNA microarrays, using electrochemical sensors and
impedance techniques in nanoscale pores (51 refs) (138). Wang
wrote a review on nanoparticle-based electrochemical DNA
detection, including his own work in this area (18 citations) (139).
The group of Willner reviewed the use of magnetic particles for
the development of biosensors as well as electrochemical DNA
and immunoassays (41 refs) (140). Mascini’s group reviewed the
fabrication and selection methods of aptamers and the use of these
artificial nucleic acid ligands as affinity biocomponents in biosen-
sors (so-called aptasensors), with 50 references (141). The group
of Willner also wrote a detailed review on the use of impedance
spectroscopy as a tool to probe biomolecule interactions at
3292 Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
surfaces for the development and characterization of DNA sensors
and enzyme biosensors (186 citations) (142).
Enzyme Biosensors: Glucose. Glucose biosensors comprise
the most extensively studied class of enzyme biosensors because
of the relatively high durability of the enzyme, typically glucose
oxidase, and the high practical relevance of glucose determina-
tions. To solve the problem of thermal instability, the group of
Bachas used a new thermostable glucose enzyme, glucose-6-
phosphate dehydrogenase, obtained from the hyperthermophilic
bacterium Aquifex aeolicus (143). The product of the enzyme
reaction, NADH, was electrocatalytically reoxidized by a thermo-
stable osmium complex at a graphite electrode. The amperometric
biosensor response showed excellent temperature stability even
at 83 °C and forms a highly promising addition to modern glucose
biosensor development. Most researchers in the field of electro-
chemical glucose biosensor development are targeting improve-
ments in selectivity by design of the underlying sensing material.
Electrochemical control of the entire deposition process has been
a notable development. Parallel efforts by the groups of Wilson
(144) and Schuhmann (145) found that glucose oxidase can be
electrochemically deposited by inducing a change in the local pH
at the electrode surface, which changes enzyme solubility.
Wilson’s group studied the influence of added surfactant on the
thickness of formed enzyme layer by this mechanism (144). Based
on this work, the group of Wilson reported on the electrochemi-
cally controlled deposition of a permselective layer of polyphenol
after such an enzyme deposition, which was additionally protected
by a (3-aminopropyl)trimethoxysilane membrane fabricated by
electrochemically assisted cross-linking (146). This yielded du-
rable glucose sensors with rapid response times, high sensitivity,
and low interference from undesired electroactive species. On the
other hand, Karyakin et al. used glucose oxidase embedded into
Nafion membranes from a water-organic solvent mixture in order
to stabilize the enzyme by a membrane-forming polyelectrolyte
(147). When the enzyme/Nafion casting solution was applied onto
Prussian Blue-modified glassy carbon electrodes (for improved
hydrogen peroxide response), good sensitivity toward glucose
sensing was observed.
Other authors continued work on the electrical wiring between
glucose oxidase and the underlying electrode for efficient electron
transfer. The group of Willner covalently attached N-6-(2-amino-
ethyl)flavin adenine dinucleotide as a linker between glucose
oxidase and a redox polymer composite polyaniline/poly(acrylic
acid) (148). This direct electrical contact yielded very high
electron-transfer rates. The mechanism of such a glucose sensing
architecture was studied by in situ surface plasmon resonance,
and the sample glucose concentration was shown to control the
steady-state concentration ratio of reduced and oxidized form of
polyaniline. The group of Watanabe studied a series of pheno-
thiazine-labeled poly(ethylene oxide) linked to lysine residues on
glucose oxidase as electrical wires for glucose sensing (149). A
maximum catalytic current was observed for a linker size of 3000
Da. Palmisano et al. used a composite of tetrathiafulvalene-
tetracyanoquinodimethane crystals and overoxidized polypyrrole,
giving a direct electrical connection between enzyme and under-
lying platinum electrode (150). Efforts also continued in direction
of miniaturization. Hrapovic and Luong, for example, fabricated a
glucose biosensor with tip diameters estimated between 10 and
500 nm (151). The enzyme was entrapped by electropolymerized
phenol and 2-allylphenol, similar to the systems discussed above.
Novel concepts for electrochemical glucose sensing were also
reported. Tlili et al. used fibroblast cells grown on an optically
transparent indium tin oxide electrode (152). They found that the
electrochemical impedance response changed reproducibly with
the glucose concentration in the sample in the range of 0-14 mM,
with other sugars showing no interference.
Other Enzyme Biosensors. Site-directed mutagenesis was
used by Bao et al. to fabricate an amperometric histamine sensor
with improved detection limits (153). For this purpose, phenyl-
alanine 55 on a subunit of the enzyme methylamine dehydroge-
nase was replaced by alanine, giving a 400-fold lower Km value in
solution and a 3-fold lower value when immobilized into a
polypyrrole sensing matrix. The resulting detection limits were
found to be 4-fold lower for sensors with the modified enzyme.
The group of Hall used site-specific mutations on trimethylamine
dehydrogenase to facilitate electrical wiring between enzyme and
redox mediators at the electrode (154). Two different mutants
were designed and studied in detail, and the most promising
enzyme was successfully immobilized into an electrochemical
sensor configuration where direct electrical wiring to an iron-based
redox polymer was confirmed electrochemically. In analogy to
their glucose work cited above, the group of Bachas developed
an improved biosensor for asparagine on the basis of a thermo-
stable recombinant asparaginase (155). The enzyme was found
to be thermostable up to 85 °C in solution and was placed in front
of an ammonium-selective electrode to fabricate a potentiometric
sensor with a 6 × 10-5 M detection limit for L-asparagine.
Naal et al. fabricated an amperometric sensor for the explosive
2,4,6-trinitriotoluene (TNT) on the basis of the oriented im-
mobilization of a nitroreductase maltose binding protein fusion
(156). In contrast to the immobilized fusion protein, the wild-type
nitroreductase alone lost most of its enzymatic activity when
deposited onto the electrode modified with an electropolymerized
film. Detection limits for TNT were ∼2 µM.
Aoki et al. continued their work on silicon-based light addres-
sable pH electrodes, where only the illuminated microdomain
gives rise to a potentiometric response, to the fabrication of an
enzyme-based multianalyte sensor for sucrose, maltose, and
glucose (157). Different spots on the chip were coated with
appropriate thermophilic enzymes for improved durability and
illuminated with light-emitting diodes. Chemometric analysis of
the results was explored for better accuracy.
Numerous papers continued to use various polyelectrolytes
(polymers and clays) to stabilize enzymes in biosensor configura-
tions. For example, Kanungo et al. entrapped enzymes into poly-
(styrenesulfonate)-polyaniline composites that were synthesized
within the pores of track-etched polycarbonate membranes, which
resulted in immobilizing the enzymes during polymerization (158).
Compared to classical polyaniline-based biosensors, an increase
in linear response range and a decreased response time was
observed. A microtubule sensor array was constructed on the basis
of this principle for the simultaneous measurement of glucose,
urea, and triglyceride in the same sample. In another example,
Wei et al. used the polycationic biopolymer chitosan to form thin
biopolymer films containing the polyanionic enzyme lactate
oxidase (159). A much improved stability of the enzyme was
found, and lactate sensors with detection limits down to 50 nM
were constructed. Yu et al. reported on an efficient electrical wiring
of enzymes for biosensor construction (160). A 4-nm layer of
sulfonated polyaniline on a polycationic underlayer was covered
with a film containing the enzyme (myoglobin or horseradish
peroxidase) and poly(styrenesulfonate). It was shown that 90%
or more of the protein was electrically coupled to the electrode,
giving an improved biosensor sensitivity with a 3 nM detection
limit for hydrogen peroxide.
While carbon paste has been found to be an attractive matrix
for biosensor research because it can be doped with catalysts and
biomolecules, Mailley significantly improved such amperometric
biosensors by using a composite of carbon paste and in situ-
generated polypyrrole containing the enzyme polyphenol oxidase
for catechol detection (161). The composite exhibited much
improved enzyme retention because of its effective entrapment
by polypyrrole.
Abad et al. introduced an immobilization technique to attach
glycosylated proteins covalently to self-assembled monolayers on
gold electrodes (162). Rather than using boronic acids, which
form reversible bonds with saccharides, the authors combined
such boronates with epoxy groups to achieve a very stable
covalent linkage.
A method to determine the concentration and isomer ratio of
urocanic acid, which is important in understanding the photo-
immunosupression in the skin, was developed by Tatsuma et al.
by monitoring the inhibition of the hydrogen peroxide reduction
at a heme peptide-modified electrode (163). Since the two different
isomers show different inhibition of this response, the current
before and after UV irradiation, which transforms the trans into
the cis isomer, could be used to estimate the isomer ratio of the
analyte.
Mao et al. developed an enzyme-modified ring-disk carbon
film electrode embedded in a thin-layer radial flow cell for the
determination of trace amounts of hydrogen peroxide from brain
microdialysate (164). While the ring electrode contained horse-
radish peroxidase for actual hydrogen peroxide detection, the disk
electrode contained ascorbate oxidase to preoxidize and eliminate
ascorbic acid that would otherwise interfere with the on-line
analysis. In an alternate approach, Choi et al. explored the use of
an insoluble oxidant membrane placed in front of an enzyme
containing film to remove interfering oxidizable species (165).
Creatinine and glucose biosensors were used as model systems,
and the best oxidant was determined to be PbO2.
Rather than using enzymes as biocatalysts to detect their
substrates, Neufeld et al. electrochemically determined the
enzymes released from lysed bacteria as a method to quantify
and identify bacteria (166). In this example, a bacteriophage
specific for Escherichia coli was used to release the bacterial cell
content into solution. Amperometric detection of the marker
enzyme activity (a galactosidase) gave detection limits as low as
1 colony-forming unit/100 mL sample.
Sun and Jin determined zeptomole quantities of enzymes from
individual human erythrocytes by electrokinetically injecting the
sample into a capillary where the sample was electromigrated to
a region of higher temperature to initiate enzyme reaction (167).
The electroactive product NADH of the model enzyme glucose-
6-phosphate dehydrogenase used here was then monitored at a
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004 3293
carbon fiber disk bundle electrode. This is one of many examples
of a hyphenated technique where electrochemistry is used to
inject, separate, and detect the analyte.
Immunosensors. The principles of electrochemical immuno-
sensors are now well established, and current developments go
mainly in the direction of miniaturization and the fabrication of
array systems in the form of biosensor chips and the exploration
of alternate interrogation principles. The group of Fritsch reported
on an immunoassay in a microcavity format containing a recessed
microdisk with covalently attached antibody and a nanoband gold
electrode for voltammetric detection of the enzyme label reaction
product, p-aminophenol, with 56-zmol detection limits for the
detection of IgG (168). In another example, Kojima et al.
developed an electrochemical protein chip with an array of 36
platinum electrodes, in addition to thin-film silver/silver chloride
electrodes and auxiliary electrodes, integrated on a glass substrate
(169). Immobilization was achieved by plasma polymerization of
a siloxane structure that showed no detectable nonspecific
adsorption, and independent enzyme labeled sandwich immu-
noassays were successfully performed at different sites on the
chip.
The group of Smyth developed a competitive electrochemical
enzyme-labeled immunoassay for sequential analyses of atrazine
without any washing or regeneration steps (170). This was
achieved by allowing the redox centers of the horseradish
peroxidase enzyme label to couple directly to the conducting
polymer substrate. Atrazine was detected down to 0.1 ppb
concentrations.
Grant et al. improved on an interesting label-free and reversible
electrochemical immunosensor principle originally reported by
Sadik and Wallace (171). The antibodies (against bovine serum
albumin and digoxin) were embedded into conducting polypyrrole
films and interrogated by pulsed amperometry. The chrono-
amperometric responses were reversible in quiescent solutions
and showed a linear measuring range between 0 and 50 ppm.
Dai et al. proposed the use of a pseudoreagentless ampero-
metric immunosensor based on the direct electrochemistry of
horseradish peroxidase (172). This enzyme was labeled to the
antibody for the target antigen (carcinoma antigen-125), which
were both deposited onto the sensor platform before measure-
ment. When increasing concentrations of antigen were present
in the sample, the current from the enzyme was found to decrease
because of the competition between antigen present in the sample
and immobilized on the electrode surface, yielding an apparently
reagentless assay.
Capacitance and impedance techniques are increasingly being
used to probe immunoreactions at electrode surfaces, somewhat
in analogy to surface plasmon resonance. Of course, as a label-
free technique, they are potentially very versatile but more prone
to effects from nonspecific adsorption than established voltam-
metric techniques using an enzyme label, for example. The group
of Sadik used differential impedance spectroscopy to monitor the
kinetics and surface loading of protein immobilization and
antibody-antigen reactions as a fundamental technique to un-
derstand surface deposition mechanisms and surface reactivity
(173). Corry et al. also probed antibody-antigen binding events
at gold-coated quartz crystals and indium-doped tin oxide films
by electrochemical impedance spectroscopy (174). The electro-
3294 Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
chemical results were compared to surface roughness experiments
using atomic force microscopy. Zayats et al. used impedance
measurements on ion-sensitive field-effect transistor devices to
determine the film thicknesses of the biomaterial, with good
correlations to surface plasmon resonance measurements on the
same system (175). This device is mainly useful for the detection
of large analytes such as antibodies or the toxin cholera, for which
an assay was developed.
Oligonucleotides: Direct Detection. Numerous label-free
detection methods have been explored in the past few years. De
los Santos-Alvarez electrochemically oxidized the adenine bases
of adsorbed oligonucleotides on pyrolytic graphite electrodes
(176). They found that the reaction products were electroactive
and strongly adsorbed onto the electrodes, which could be used
to detect specific DNA sequences and synthetic homopolynucle-
otides. In a similar approach, Jelen et al. treated DNA with a strong
acid to release its purine bases that were then detected by cathodic
stripping voltammetry on a copper amalgam or hanging mercury
drop electrode with subnanomolar detection limits (177). Ozkan
et al. used the direct electrochemical oxidation of the guanine
bases by differential pulse voltammetry at a carbon paste electrode
to monitor the hybridization of DNA (178). Since peak currents
for this assay were different for an allele-specific mutation, a label-
free yes/no system for the desired mutation was developed. The
group of Mascini also used the electrochemical response of the
guanine bases in the target DNA in a label-free assay but
substituted all guanine bases in the immobilized probe DNA by
inosine (179). This assay was developed in view of the detection
of PCR samples of 244-base pair fragments related to the
apolipoprotein E in just 10 min. Palecek’s group significantly
reduced the problem of nonspecific adsorption of undesired
nucleotides at the electrode surface by physically separating the
recognition and detection surfaces (180). Hybridization was
achieved at paramagnetic beads, followed by acid treatment that
released adenine into solution that was detected at a mercury
electrode. This sensitive label-free method was demonstrated with
numerous types of oligonucleotides, and some possibilities to
further increase the sensitivity through the use of catalytic
schemes were discussed. See also Janata’s work above for yet
another example of a label-free DNA detection method (67).
Oligonucleotides: Intercalator Detection. The electro-
chemical detection of DNA via redox-active or electrocatalytic
intercalators is an attractive approach to oligonucleotide hybridiza-
tion measurements because the target DNA does not need to be
chemically modified. For example, Maruyama et al. developed an
osmium(II) complex containing amine electron-donating groups
that showed a high binding affinity (3 × 107 M-1) to double
stranded DNA and a low half-wave potential (181). When probe
DNA was immobilized onto a gold electrode, detection limits for
the electrochemical determination of target DNA was found to
be 0.1 ng L-1 with a wide linear range. In a related approach,
albeit not with a classical intercalator as reporter molecule,
Masarik et al. proposed the adsorptive transfer stripping square
wave voltammetric detection of streptavidin and avidin to quantify
DNA hybridizations of biotinylated oligonucleotides (182). Detec-
tion limits were found to be as low as 6 pM for denatured
streptavidin. Homberg and Thorp performed an electrochemical
study and digital simulation to quantify the binding and rate
constants for the reaction of DNA with two different intercalators
used simultaneously, one acting as an electrocatalyst for guanine
oxidation, giving higher currents with higher double-stranded
DNA concentrations, and the other used as redox probe, giving
lower currents in the presence of DNA because of decreased mass
transport (183). Wong and Gooding explored a mixed monolayer
on gold containing single-stranded DNA and incubated with the
redox-active intercalator 2,6-disulfonic acid anthraquinone for DNA
detection (184). Only when complementary DNA was allowed to
interact with the monolayer were voltammetric peaks for the
oxidation and reduction of the intercalator observed, indicating
that the double-stranded DNA was needed for electron transfer.
Binding to DNA with mismatched base pairs gave reduced signals.
Yang et al. used the polymerase chain reaction to amplify the
desired DNA with 7-deaza analogues of guanine and adenine in
order to obtain a larger electrochemical oxidation current in the
presence of a ruthenium(II) bipyridine as electrocatalyst (185).
Fahlman and Sen proposed molecular design strategies in order
to use the change in electron-transfer properties of double-
stranded DNA as an aptamer for the detection of intercalators,
not the other way around (186). However, the selectivity of such
a sensor was not yet characterized and is perhaps quite limited.
Mugweru and Rusling developed a self-contained probe for
damaged DNA with a catalytic film containing the DNA inter-
calator ruthenium-bipyridine and square wave voltammetric
detection (187). When double-stranded DNA was subjected to the
suspected carcinogen styrene oxide, the catalytic current was
found to increase linearly with time. The mechanism of this assay
was explained with the catalyst having improved access to the
oxidizable bases of the damaged and partly unwound DNA,
thereby increasing the current compared to undamaged DNA. In
a related approach, Zhou et al. screened for DNA damage by
forming a multilayer thin film containing the double-stranded DNA
of interest and myoglobin or cytochrome P450 (188). Upon
activation with acid, sample styrene was converted to the carci-
nogenic styrene oxide in situ by the enzyme and the intercalators
ruthenium(II) and cobalt(II) bipyridine were used to electro-
chemically distinguish between intact and damaged DNA. Kelley’s
group used the electrocatalytically enhanced voltammetric ruthe-
nium(III) hexamine response to monitor DNA hybridization at a
gold electrode (189). Since the ruthenium complex interacts
electrostatically with DNA, it leads to a larger current when
hybridized DNA is present. A single base pair mismatch could
be identified by following the voltammetric response as a function
of hybridization time. Yamashita et al. found in a detailed study
that single base pair mismatches in DNA assays involving a 20-
mer probe can be electrochemically identified by using the
intercalator ferrocenylnaphthalene diimide (190). Quartz crystal
microbalance and MALDI-TOFLMS studies confirmed that the
number of binding intercalator molecules decreased with increas-
ing number of base pair mismatches.
Oligonucleotides: Enzyme Amplified. Enzyme-amplified
electrochemical oligonucleotide assays have been developed in
analogy to earlier enzyme-labeled immunoassays. Aguilar and
Fritsch introduced such an adaptation to a classical sandwich assay
to detect Cryptosporidium parvum in water samples (191). The
probe DNA was attached via its 5′-amine terminus to a self-
assembled monolayer of mercaptoundecanoic acid on a gold
electrode. Upon hybridization with the target 121-nucleotide
sequence, a secondary DNA probe tagged to alkaline phosphatase
was hybridized. The enzyme generated aminophenyl phosphatase,
from its added substrate p-aminophenol, which was detected
electrochemically at the gold electrode. Detection limits were
found to be ∼150 nM, with good selectivity. Kim et al. used the
same enzyme reaction in a related DNA assay, but by using an
aminated dendrimer containing ferrocenyl groups as electrocata-
lyst between the self-assembled monolayer and the DNA probe
to increase sensitivity (192).
The group of Heller used a carbon electrode chemically
modified with a redox polymer and electrodeposited avidin to
construct a sandwich assay, with probe DNA or RNA binding to
the target, which in turn binds to an enzyme-labeled oligonucle-
otide delivered to the sample (193). The electrode was made
specific by conjugating biotinylated probe RNA or DNA to the
deposited avidin. Upon cohybridization with the target oligonucle-
otide and a horseradish peroxidase-tagged oligonucleotide, a
sandwich was formed that was interrogated electrochemically by
measuring the hydrogen peroxide reduction current. The electrical
wiring of the enzyme with the redox polymer, which this group
has already successfully used for glucose sensor development, is
one of the key features of this electrochemical assay that takes
∼30 min to complete. Subsequent work of the same group used
a microelectrode configuration for increased mass transport and
achieved a 100-fold improvement in detection limit down to ∼20
pM levels (194). Williams et al. developed a related method for
rapid DNA screening, using a very simple modified streptavidin
carbon-polymer composite electrode that can be renewed by
polishing between measurements (195). In this approach, target
and probe DNA and a horseradish peroxidase enzyme label bound
to a suitable antigen are all added to the sample at the same time,
eliminating separate binding and washing steps.
Willner’s group utilized an enzyme label for DNA detection
that produces an insoluble reaction product (196). The readout
was accomplished by impedance spectroscopy (and by a quartz
crystal microbalance), and DNA detection limits were found to
be on the order of 10-13 M.
Oligonucleotides: Nanoparticles and Quantum Dots.
Nanoparticle labels for oligonucleotides are known to be very
attractive in spectroscopic readout methods and share unique
properties that are very useful for electrochemical detection as
well. In many cases, metal nanoparticles can be oxidized to form
metal ions that are conveniently determined electrochemically. A
recent example for this approach was described by Oxsoz et al.,
who monitored the direct oxidation current of gold nanoparticle
tags upon hybridization of tagged target DNA and probe DNA
covalently attached onto a graphite electrode (197). In another
approach, the group of Wang used gold nanoparticles coated with
ferrocenylhexanethiol and streptavidin (the latter for attachment
of the biotinylated DNA probe) (198). Upon forming of a DNA
sandwich, the ferrocene groups were detected electrochemically
with a linear measuring range for DNA between 7 and 150 pM.
The main advantage of this method lies in its experimental
simplicity since no enzyme or enzyme substrate is needed and
amplification is achieved by the large number of ferrocene groups
present for each DNA binding event.
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004 3295
 Recently, such nanoparticle tags have been combined with
magnetic particles for the purpose of additional preconcentration
(199). Here, magnetic particles with probe DNA were used to
capture target DNA, which in turn was allowed to hybridize with
a secondary probe DNA tagged to a given metal nanoparticle. After
hybridization, the ensembles were preconcentrated magnetically
at an electrode and the nanoparticles were oxidized chemically
and detected by anodic stripping voltammetry. The authors
demonstrated simultaneous DNA assays with 0.3 nM detection
limits by introducing up to three different nanoparticle tags (ZnS,
CdS, and PbS) that could easily be electrochemically resolved.
The same authors also introduced polystyrene beads containing
defined amounts of various nanoparticles as electrochemical
encoded tags in complete analogy to fluorescent polystyrene tags
used in flow cytometry or random fiber-optic arrays (200). The
electrochemical signatures were found to correlate well with the
original nanoparticle loading concentrations.
CONCLUSIONS
The topic of electrochemical sensors is already quite vast and
continues to grow and broaden. The field of potentiometric
sensors, as a mature technology, has experienced important
change in the past few years. The principal developments in this
area focus on reducing the detection limit to true trace levels,
down to the low parts per trillion concentration range, and there
are important advances in the areas of materials and active
components design. Importantly, potentiometry and the field of
ion-transfer voltammetry start to approach each other to the extent
that the design of instrumentally controlled ion-selective electrodes
now becomes possible. Voltammetric sensor development focuses
on further miniaturization, the reduction of the addressable sample
volume, and the application to difficult in vivo and environmental
sensing situations. Moreover, numerous materials characteristics
are being improved to achieve improved selectivity as well as a
larger potential window in aqueous samples. Electrochemical gas
sensors are based on a wide range of mechanisms, ranging from
simple resistance measurements to true electrochemical conver-
sions at a three-phase interface. Developments in this area are
quite divergent, with some researchers targeting the direct
selective detection of analytes based on materials properties as
well as the magnitude of the applied potential, others using an
array of simpler, less selective systems in conjunction with a
separation device such as a portable gas chromatograph, and yet
others pursuing the concept of the electronic nose with an array
of rather nonspecific sensors. It must be noted that the last concept
has been rather successful for the distinction of individual gases
or, at the most ternary mixtures, but normally fail at analyzing
complex sample mixtures as they are often encountered in the
real world.
Electrochemical biosensor concepts are a vast area of research
that continues to develop at a rapid pace. The enzyme-based
biosensor is the classical biosensor, and the development of the
glucose sensor is still the largest area of research, although it is
very often used as a model system. Thermophilic enzymes for
higher stability, improved materials for better biocompatibility,
reduced interference, and improved enzyme stability, and the
electronic wiring of enzymes to electrodes for mild and direct
transduction of the signal are all important approaches that have
3296 Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
been explored. Affinity sensors using DNA or immunological
recognition units are perhaps not classical sensors because they
lack in many cases reversibility. Yet, the number of electrochemi-
cal detection schemes for measuring these extremely important
analytes is very inspiring. They range from direct label-free
detection principles or intercalator-based techniques, the use of
enzyme labels that form electroactive or insoluble products, to
the application of quantum dots and magnetic particles as labels.
In many of these detection principles, extremely low levels of
detection with excellent selectivity and the capability of detecting
single base pair mismatches has been demonstrated.
Clearly, the area of electrochemical sensor research is very
active and fruitful. It must be emphasized that many of the
challenges that remain in some cases, especially in the area of
selectivity, may be overcome by their integration into more
complex analytical systems that combine online sampling and
separation steps. However, in the cases where direct detection in
unmodified samples is possible, the high analysis speed and the
capability of detecting extremely small volumes without signifi-
cantly perturbing the sample remain highly attractive character-
istics of electrochemical sensors.
ACKNOWLEDGMENT
This author gratefully acknowledges the National Institutes of
Health and the Petroleum Research Fund (administered by the
American Chemical Society) for supporting his research on electro-
chemical sensors.
Eric Bakker is currently an Alumni Professor in the Department of
Chemistry at Auburn University. After undergraduate and graduate studies
of chemistry and analytical chemistry with the late Wilhelm Simon at the
Swiss Federal Institute of Technology in Zurich, Switzerland, he pursued
postdoctoral studies at the University of Michigan. He joined the faculty
at Auburn University in 1995 as an Assistant Professor and was promoted
to Associate Professor in 1998 and to full professor in 2003. His research
interests include fundamental and applied aspects of potentiometric,
voltammetric, and optical sensors based on molecular recognition and
extraction principles. He has published about 120 papers in this field.
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