Anal. Chem. 2004, 76, 3285-3298 Electrochemical Sensors Eric Bakker
Department of Chemistry, Auburn University, Auburn, Alabama 36849
sensors, even though they are classified as microscopic tech-
niques. Hyphenated systems, such as microdialysis probes
coupled to an electrochemical detection system, optionally after
an on-line separation step, act as sensors as well. The reader of
this fundamental review must therefore keep in mind that
integration between fields is at an advanced stage and many
exciting developments cannot be discussed here because of space
limitations and overlap to other topics discussed separately.
A 200 reference limit was used, which means that only a
fraction of relevant works are covered here. This review attempts
to present a relatively good coverage of review articles and a
Sensors Based on Electrochemically Driven
selection of original research articles that emphasize new chemical
developments or principles as opposed to solving analytical
problems. The ACS SciFinder and, to a lesser extent, the ISI
Science Citation Index were used to compile the selection of papers
presented here. Subject and author searches were performed, and
for a number of key journals, the table of contents listing was
manually read. Only journal articles written in English were
considered. The patent literature and conference proceedings or
abstracts were ignored. Electrochemical sensors comprise the
largest group of chemical sensors. Because of the comparatively
large number of review articles that have been published and the
breadth of this research topic, this review will not be able to give
due credit to all the excellent work that is being done in this field.
Oligonucleotides: Nanoparticles and Quantum
This author therefore apologizes to anybody who feels that some
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
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
230 references (6). Such polymers are primarily used as inner
Detection Limit. The discovery that the detection limit of
reference elements, but have been explored as ion-selective
ionophore-based ISEs can be lowered to trace levels has initiated
interesting research by a number of groups. A steady-state model
Theory and Characterization. A detailed extension of earlier
was developed to predict the lower detection limit of ionophore-
work on the theory of so-called non-Nernstian equilibrium
based ISEs (14). Importantly, this model allows the experimental-
responses of ionophore-based ISEs was presented by Amemiya
ist to calculate optimal inner solution compositions so that rational
et al. (7) Non-Nernstian slopes based on zero current sample
design becomes possible. Experiments with silver-selective mem-
concentration polarizations, which are important for low detection
branes correlated well with predictions when an ion-exchange
limit ISEs, were not discussed here. As long as the ionophore
resin was used to buffer the inner solution, to avoid undesired/
complexes primary and secondary ions independently, the model
unknown extraction processes from the inner membrane side. The
shows how the observed electrode slope must change as a
detection limit of anion-selective electrodes was also evaluated
function of the charges of ions and ionophore involved. This
(15). It was found that the inner solution plays a minor role with
elegant work has important implications for future ionophore and
ionophore-free membranes and that the detection limit of an
iodide-selective electrodes based on the mercuracarborand-9
The determination of stability constants of ion-ionophore
ionophore could be lowered to nanomolar levels. In all cases,
complexes directly in ion-selective membranes has been a chal-
hydroxide was found to be the primary sample interference that
lenge for many years. Among the methods recently introduced,
dictates the detection limit via counterdiffusion fluxes. Michalska
the so-called sandwich membrane method, which utilizes a
et al. have published papers on lowering the detection limits of
concentration polarized membrane made up of two segments of
ISE with a conducting polymer solid contact (16). The earlier
known composition, is one of the most powerful. Mikhelson et
water layer theory of Pretsch was not considered here. Rather, it
al. have made their early work, published in Russian, more
was argued that the self-discharge of the conducting polymer
accessible and discussed the method in depth, focusing on their
caused the very high detection limit ordinarily observed with such
titration technique, the determination of complex stoichiometries,
systems and that an anodic coulometric control could be used to
and estimation of possible biases such as ion pair formation and
reduce the detection limit by ∼3 orders of magnitude to submi-
diffusion potential (8). Among other papers, this method was
cromolar levels. The observed detection limits are still much
utilized by Ceresa et al. to characterize the binding properties of
higher than that observed with optimized liquid contacts, however.
a highly halide selective ionophore, mercuracarborand-3 (9),
The group of Pretsch found that the use of microspheres normally
which had been introduced earlier by Bachas and Hawthorne.
used in HPLC, placed on the sample side of ISE membranes,
The complex formation constants observed for this ionophore
completely eliminated undesired super-Nernstian response slopes
corresponded quantitatively to extraction constants determined
to give subnanomolar detection limits for calcium (17). Pretsch
with optical sensors based on the same ionophore, showing once
also used a rotating electrode configuration, with the membrane
more that diffusion potentials are generally not significant with
placed off-center of the rotating axis, to yield detection limits in
ion-selective membranes. The protocols for determining complex
the picomolar range and rapid response times (18). In other work,
formation constants by this method were extended to the
an electrode rotator was used as a diagnostic tool to evaluate the
quantification of membrane acid dissociation constants of H+-
level of optimization of the ISE in terms of its detection limit (19).
selective ionophores and formation constants for electrically
De Marco’s group studied the effect of diffusion fluxes on the
charged anion ionophores (10). The sandwich method was also
detection limit of the commercially available jalpaite copper ion-
found to be useful for the quantification of electrolyte coextraction
selective electrode (20). Indeed, rotating disk electrode experi-
processes (11). When one membrane segment contained a cation
ments revealed that the detection limit could be lowered to about
exchanger and the other an anion exchanger, the observed
nanomolar levels. The detection limit could be further decreased
potential of the combined sandwich could be related to the
by using modified membrane compositions to further minimize
coextraction constant of the respective electrolyte, which in turn
copper dissolution, using an excess of sodium sulfide.
was used to successfully predict the upper detection limit of
Component Design. Polymer membrane-based ISEs require
ion-exchanger properties, and historically, tetraphenylborate de-
While the sandwich membrane method mentioned above has
rivatives have been incorporated for cation-selective electrodes.
been used earlier for the determination of ionic impurities in ISE
Unfortunately, they have limited chemical stability and lipophilicity
membranes, Gyurcsanyi and Lindner have used an optical method
and are very difficult to covalently anchor onto the polymeric
to do the same, where the level of protonation of a dilute
backbone. In response to this problem, perhalogenated closo-
electrically neutral lipophilic pH indicator in the membrane is
dodecacarboranes have been introduced as alternative compounds
quantified (12). This method works when ion-exchange and
in ISE membranes, with characteristics that equal or surpass that
electrolyte coextraction processes are experimentally excluded.
of the best available tetraphenylborates (21). In subsequent work,
De Marco and co-workers continued to apply an array of
this class of compounds was covalently attached onto a meth-
surface characterization techniques to the understanding of ion-
acrylic copolymer, and the ion selectivity of the membrane was
selective electrodes. In one example, the fouling of mercury(II)
perfectly retained (22). On the basis of this ion exchanger, a
chalcogenide electrodes upon prolonged contact in saline solution
calcium-selective membrane based on a plasticizer-free polymer,
was studied by XPS, SIMS, RDE-EIS, and SR-GIXRD, and it was
with chemically attached calcium ionophore and cation exchanger,
found that fouling is linked to the poisoning by silver salts (13).
was successfully fabricated for the first time (22).
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
It is known that calcium-selective ionophores form very stable
IR spectroscopy and X-ray diffraction) went along with significantly
complexes in the membrane. For this reason, the resulting
enhanced ion selectivity, which they explained with cooperative
membranes are often subject to electrolyte coextraction at elevated
interaction of adjacent crown ethers.
concentrations, which leads to anion interference. The group of
Polymeric membranes modified with Zeolite particles were
Nam has introduced a new tweezer-type ionophore with much
used by the group of Walcarius for the preparation of ammonium-
lower complex formation constants (23). While the resulting
selective ISFETs (31). While a very high Zeolite content was
selectivity is somewhat inferior to that of the best available calcium
needed (43 wt %) and the observed electrodes slopes were
ionophores, anion interference was completely eliminated, even
severely sub-Nernstian, a low detection limit of 10 nM was
in calcium perchlorate solutions up to 0.1 M. Similarly, a new
polymerizable derivative of ETH 129 was shown to form weaker
An anodically electrodeposited iridium oxide pH microelec-
complexes than its parent molecule ETH 129 (24). Membranes
trode was fabricated and characterized by Bezbaruah and Zhang
containing the covalent attached calcium ionophore showed
(32). While response times were fast and the pH measuring range
somewhat inferior selectivity as well, but a reduced anion interfer-
was large, interference from redox-active compounds was signifi-
ence. Importantly, the covalent anchoring of this ionophore
cant. Yamamoto et al. fabricated a tungsten nanoelectrode (with
resulted in Nernstian response slopes, even with a calcium-free
400-800-nm tip diameters) by etching a tungsten wire followed
inner solution that would otherwise induce a strong inward ion
by electrooxidation to tungsten oxide (33). The electrode, which
flux. This was attributed to the reduced calcium mobility in the
gave a measuring range between pH 2 and 12, was applied to
extracellular pH measurements on endothelial cells.
Different molecular design strategies were used by Sasaki et
The group of Bachas used an ISE membrane covered with a
al. for the recognition of the ammonium ion (25). A tripodal
polymer containing phosphorylcholine functionalities, which mimic
preorganization to reject the spherical potassium was successful,
the polar groups on cell surfaces (34). Decreased adhesion and
but the resulting membrane suffered from calcium interference.
activation of platelets was demonstrated by immunostaining, and
The use of crown ethers containing bulky decalino blocking units
ISE sensing characteristics were not affected by the coating. See
gave the best results, with membranes that have better ammonium
also Voltammetric Sensors for other approaches to biocompat-
selectivity than any system reported to date.
Uranyl salophenes have been established as very promising
A novel potentiometric measuring principle for the detection
phosphate ionophores but have traditionally suffered from chemi-
of saccharides was developed on the basis of poly(aniline boronic
cal decomposition in contact with phosphate solutions. Wojciechows-
acid) (35). The complexation resulted in a change of the effective
ki et al. have used a modified inner solution, in analogy to work
pKa of the polymer, which gave rise to a change in the observed
on low detection limit ISEs, that effectively buffers both phosphate
potential. Additives such as Nafion or sodium fluoride also had a
marked effect on sensor sensitivity. 26). The resulting ion flux in the direction of
the inner solution keeps the membrane mostly phosphate-free,
van der Wal et al. introduced a technique for the simple
covalent attachment of poly(vinyl chloride) membranes to solid
thereby increasing the lifetime of such membranes to over two
substrates such as glass and other oxide surfaces (36). Mem-
branes attached via a silane coupling agent containing an amine
Bobacka and co-workers explored the recognition of aromatic
group and a heating step showed excellent adhesion properties
cations such as N-methylpyridinium based on π-coordinating
and retained their sensing characteristics relative to membranes
carriers that were either electrically charged or neutral, in
cast without linking agent. This work potentially solves a long-
membranes containing different plasticizers and backside con-
standing problem in the development of ion-selective field effect
tacted with a polythiophene-type solid contact (27). Only electri-
transistors, without the need for a new polymeric material.
cally charged carriers were found to show significant changes inselectivity. REFERENCE ELECTRODES
The group of Nam has assessed so-called tweezer-type carbon-
Very few works reported on new reference electrode principles.
ate ionophores to the measurement of carbon dioxide in seawater
Langmaier and Samec explored freshly polished copper wires
(28). With one of the ionophores, a very high carbonate selectivity
directly inserted into an electrolyte solution of the solvent
over chloride (log Kpot, -6) and other minor ions was observed,
o-nitrophenyl octyl ether as a junction-free inner reference
sufficient for direct determination in seawater without sample
electrode in ion-transfer voltammetry (see topic below) (37). They
pretreatment. A comparison of seawater analyses to reference
reported impressive stabilities on the order of 2 mV over 300 h,
but the exact mechanism of this behavior could not be explained.
Malinowska et al. studied zirconium(IV)porphyrins as electri-
Lee and Sohn explored field effect transistor-type reference
cally charged ionophores for fluoride (29), which was preferred
electrodes consisting of a one ISFET gate covered with a pH-
over all other tested ions, including the lipophilic perchlorate. A
insensitive polymer double layer that was used in conjunction with
super-Nernstian response slope was observed because of hydroxy-
a pH ISFET for pH detection (38). The mechanism for its
bridged porphyrin dimer formation as confirmed by UV/visible
functioning remains unclear as well. Membrane Materials. Kimura et al. have continued to study VOLTAMMETRIC SENSORS
their thermotropic liquid crystals as ion-selective membranes
Reviews. Wang reviewed the current status and future
doped with crown ether ionophores (30). They found that an
challenges of miniaturizing electroanalytical systems, their incor-
ordered arrangement of the ionophore (confirmed by polarized
poration into microfluidic devices, and their application to point
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
of care and environmental analysis as well as genetic testing, with
their work on the development membranes containing pores of
17 references (39). In another review, the same author stressed
molecular dimensions for new separation and electrochemical
the usefulness of electrochemical sensors for environmental
sensing applications, with 45 citations (58).
monitoring applications as an approach to a greener analytical
Interrogation Principles. The groups of Bachas and Grimes
chemistry compared to traditional instruments (35 refs) (40). The
introduced a novel measuring principle to monitor electrochemical
group of Wightman wrote an A-page article on their work on the
processes without the need for electrical connections (59). Here,
development of electrochemical sensors for the neurotransmitter
magnetoelastic alloy films were used as the working electrode in
dopamine, with 30 citations (41). Within a special issue of TrAC,
an electrochemical cell and the mass change on the electrode
Trends in Analytical Chemistry on microelectrodes and micro-
(polypyrrole deposition) was monitored via magnetic monitoring
dialysis probes for neuroanalysis, Wightman’s group also dis-
cussed characterization and validation procedures and required
Heineman continued his work on spectroelectrochemical
selectivities of such microelectrodes when used for the measure-
sensing (see also review cited above (57)), for example, by
ment in the brain (22 refs) (42). The status of development of
describing a sensor for the detection of iron(II) (60). Here, a
electrochemical arsenic sensors for environmental monitoring
Nafion film loaded with a bipyridine ligand was used to extract
applications was reviewed by Feeney and Kounaves as part of a
iron(II) and to render it strongly absorbing by complexation.
special Talanta issue on arsenic detection, including their own
Electrochemical oxidation of this complex again rendered it
work on portable systems based on microfabricated gold arrays
colorless. In another example, the stripping voltammetric detection
(35 refs) (43). The status of electrochemical sensors for oc-
of lead and cadmium was monitored spectroscopically at the same
cupational and environmental health applications was reviewed
time, with separate wavelength regions used for each of the two
by Ashley with 109 citations, with an emphasis on rugged and
metals (750 nm for lead and 400 nm for cadmium), giving added
miniature electroanalytical devices for on-site monitoring (44).
Methods such as disposable screen printing technology for the
Ekeroth used interfacial capacitance measurements to monitor
fabrication of sensors for trace metal pollutants in a variety of
the interaction of phosphate monolayers with calcium and
sample matrixes was reviewed by Honeychurch and Hart (46
magnesium ions (62). Monitoring a non-Faradaic process, as done
citations) (45). The status of electrochemical sensors for the
here, appears to be more susceptible to effects unrelated to the
detection of metal pollutants in coastal waters was reviewed by
desired molecular interaction and should be used with caution.
Achterberg and co-workers, with 17 references (46). The history
Indeed, this approach is an alternative to others who have used
and current status of electrochemical nitric oxide sensors based
voltammetric techniques with a redox marker for quantifying
on modified electrodes was reviewed by Bedioui and Villeneuve
surface recognition events. For example, Choi et al. used a
(129 citations) (47) as well as Ciszweski and Milczarek (with 32
competitive adsorption of electrochemically inactive organic
refs, in a special NO detection issue of Talanta) (48). The
molecules such as glucose with ferrocene onto a self-assembled
application of self-assembled monolayers as a bottom-up fabrica-
monolayer containing a thiolated cyclodextrin (63). The oxidative
tion principle for the realization of electrochemical sensors for
current for ferrocene was indeed reduced with higher sample
pH and inorganic and biological sensors has been reviewed by
concentrations of glucose. A critical selectivity study of this device
Gooding et al., with 168 citations (49). This paper includes a
discussion of emerging trends such as nanotubes, dendrimers,
Baca et al. coupled anodic stripping voltammetry online to
and nanoparticles for electroanalysis. Similarly, Hernandez-Santos
ICPMS to develop a hyphenated technique with high selectivity
et al. reviewed the use of metal and semiconductor nanoparticles
and sensitivity (64). It was found that the electrochemical
for use in electroanalysis (59 citations) (50), Li et al. reviewed
preconcentration gave detection limits down to sub-ppt levels,
the electrochemistry at carbon nanotube electrodes in view of
lower than possible with conventional ICPMS, and that it could
sensor and assay development (51), and Sherigara et al. reviewed
be used to eliminate matrix effects as well.
electrocatalytic properties of nanotubes and fullerenes in view of
The group of Martin explored the use of nanotube membranes
developing electrochemical sensors, with 230 citations (52). Swain
as ligand-gated ion channel mimics (65). Ion current through the
reviewed the status and future prospects of diamond science for
membrane could be switched on or off by adding hydrophobic
numerous emerging technologies including electrochemistry and
ionic species to the sample that could interact with the hydro-
electroanalysis (53). The status of the development of electro-
phobic pores of the membrane that would otherwise be insulating.
chemical sensors based on molecularly imprinted polymers was
Sensors Based on Electrochemically Driven Extraction.
reviewed by Piletsky and Turner (45 citations) (54). Similarly,
The electrochemically controlled extraction of ions into sensing
Merkoci and Alegret reviewed the use of molecularly imprinted
polymers and other water-immiscible phases is an attractive
polymers in capacitive, conductometric, voltammetric, and poten-
approach to chemical sensing that bridges the fields of polymer
tiometric sensors (28 refs) (55). The group of Guilbault sum-
membrane-based ion-selective electrodes and voltammetry at
marized the status of chemometrics for electrochemical sensors,
metal electrodes. Wu et al. used electrochemical control of
with 78 citations, by focusing on multivariate calibration, clas-
conductive polypyrrole films to extract, preconcentrate, and desorb
sification, pattern recognition, and signal processing (56). Hei-
ionic analytes, which were subsequently analyzed by flow injection
neman’s group reviewed their own work on spectroelectrochem-
analysis and mass spectrometry (66). The method was found to
ical sensing, where electrochemistry, spectroscopy, and selective
work for a variety of cations and anions and appears to be versatile.
partitioning are combined into a single sensing device for
Janata’s group used cyclic voltammetry to control the exchange
improved selectivity (43 refs) (57). The group of Martin reviewed
of chloride ions between polypyrrole and the buffer to fabricate a
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
label-free DNA hybridization detector (67). The probe DNA was
during film formation (77). In other work, Khoo and Chen
immobilized onto polypyrrole via magnesium bridging complexes,
encapsulated methylene blue into a similar sol-gel film on glassy
and the hybridization event caused a change in the voltammetric
carbon electrodes for the electrocatalytic determination of ascorbic
and uric acid (78). The simultaneous determination of these two
In other work, a new measurement protocol was introduced
analytes in human urine samples was demonstrated. Sol-gel
for ion-selective membranes that lack ion-exchanger properties
technology was also used by the group of Mandler to design an
(68). Here, current and potential pulses were alternated to control
molecularly imprinted polymer for iron(II) using a tris(2,2′-
the extraction processes of the membrane electrochemically. The
bipyridine) complex (79). However, the achieved selectivity was
resulting responses have the same look and feel as potentiometric
not satisfactory, suggesting that the recognition and detection of
membrane electrodes, but the selectivity and response features
organic molecules is currently a more successful approach with
can be tuned and even reversed, and the reversible detection of
analytes that ordinarily give irreversible sensing responses
Domenech et al. showed that Zeolite Y containing an encap-
becomes possible. As an important early example of this approach,
sulated triphenylpyrylium ion exhibits a markedly improved
the reversible detection of the polycation protamine was demon-
oxidative response to dopamine while inhibiting the oxidation of
strated for the first time (69). In parallel work, Amemiya and co-
negatively charged interferences such as ascorbate (80). A 100-
workers used cyclic voltammetry on micropipets to demonstrate
fold excess of ascorbate could be tolerated in a differential pulse
the detection of protamine (70). In a similar effort, Samec et al.
detection mode. The group of Walcarius used Zeolites to chemi-
used cyclic voltammetry for the electrochemical detection of the
cally modify carbon paste electrodes for improved electroanalytical
anticoagulant polyanion heparin (71).
properties (81). When Zeolite particles were used instead of the
The analogy of ion-transfer voltammetry to potentiometric ion-
classical mineral oil binder of carbon paste, or used as an outer
selective electrode response was also stressed by Wooster et al.,
coating, electrodes with improved responses to copper ions were
who studied microparticles containing 7,7,8,8-tetracyanoquin-
observed after an ion-exchange accumulation step.
odimethane and tetrathiafulvalene in contact with electrolyte
Mesoporous platinum electrodes possess an enlarged surface
solutions. The voltammetric waves changed as a function of the
area that enhances their catalytic properties for chemical sensing.
type and concentration of electrolyte and were explained by ion
Consequently, Evans et al. used such materials for the enhanced
incorporation processes as well (72). Long and Bakker used
detection of hydrogen peroxide (82), and Park et al. found that
normal pulse voltammetry on pH-sensitive polymer membranes,
the normally sluggish nonenzymatic glucose response was greatly
and an apparently Nernstian relationship between sample pH and
enhanced with such electrodes (83). Enhanced electrochemical
half wave potential was also observed that correlated closely with
sugar detection after HPLC separation was also reported by You
that of corresponding ion-selective electrodes (73). Spectral
et al. by the use of highly dispersed Ni nanoparticles in a carbon
imaging experiments confirmed the electrochemical results. This
film electrode, with detection limits that were at least 1 order of
work forms the precursor for the pulsed galvanostatic approach
magnitude lower than with traditional Ni electrodes (84). Zen et
mentioned above where the potential can be directly obtained from
al. used copper-plated screen-printed electrodes for the selective
detection of o-diphenols such as catechol and dopamine in the
The group of Buffle continued their work on permeation liquid
presence of m- and p-diphenols as well as ascorbic acid under
membranes as selective preconcentrators for metal speciation
very mild conditions (-0.05 V vs Ag/AgCl) (85). The enhanced
measurements by optimizing membrane and ion channel geom-
selectivity was explained by the formation of a cyclic five-
etry (74). In this approach, the membrane is a traditional transport
membered complex intermediate at the copper electrode surface.
membrane that works on the basis of zero current counterdiffusion
Copper electrodes were also used by Paixao to determine ethanol
fluxes for the transport and preconcentration of metal ions at the
amperometrically in beverages (86). The principle was incorpo-
backside of the membrane for metal ion sensing. See the topic
rated into a flow injection analysis system and used a PTFE
above of detection limits of ion-selective electrodes for similar
membrane for ethanol extraction followed by oxidation under
alkaline conditions. The comparison of the data from beverage
Rahman et al. used a hybrid between ion extraction/recogni-
analyses with gas chromatography gave excellent agreement.
tion and redox electrodes by doping a thiophene-based conducting
Boron-doped diamond electrodes continue to be adopted for
polymer, which is normally known for its electrochemically
electroanalysis because of their high stability, low background
mediated extraction properties, with EDTA (75). The polymer was
current, and wide potential window. Ferro and De Battisti reported
coated onto a glassy carbon electrode, and the metals lead(II),
on an unprecedented 5-V potential window in aqueous solutions
copper(II), and mercury(II) were deposited and subsequently
using fluorine-terminated boron-doped diamond electrodes (87).
reduced at the electrode, with detection limits in the subnanomolar
No electroanalytical applications were yet reported with this
material. Rao et al. showed that boron-doped diamond electrodes
Electrode and Coating Materials. Ultrasonic cavitation was
are improved detectors for carbamate pesticides after HPLC
used by Cordero-Rando et al. to fabricate a sol-gel graphite-based
separation, offering better electrode stability (88). If a hydrolysis
electrode material from an acidic aqueous solvent in view of
step was introduced prior to separation and detection, ppb
developing electrochemical sensors (76). The group of Collinson
detection limits were achieved for these analytes. The group of
used an electrodeposition process from a tetramethoxysilane sol
Swain explored boron-doped diamond films as electrically trans-
to fabricate sol-gel silicate films that were rougher than spin-
parent electrodes on quartz for spectroelectrochemical applica-
coated films. Various redox molecules were electroencapsulated
tions, obtained by microwave-assisted chemical vapor deposition
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
(89). The optical and electrochemical properties of the films were
range was only in the millimolar range, selectivity was clearly
found to be extremely stable, even in harsh environments, and
improved relative to an uncoated gold electrode. See the work of
found to be superior to that of traditional In-doped tin oxide thin
Rhaman et al. discussed above for other types of selective coatings
films. In analogy to carbon paste, monocrystalline diamond paste
(75). Willner’s group used imprinted membranes as coatings on
electrodes were introduced and studied by Stefan and Bairu for
field-effect transistors for the detection of triazine herbicides (99).
the determination of iron(II) in pharmaceutical preparations (90).
Although sensitivities were rather small, large selectivity changes
Wang and Musameh incorporated the electrocatalytic advan-
and reversals were obtained upon imprinting with various herbi-
tages of carbon nanotubes into a more rugged configuration by
cide substrates, making this a promising technique.
forming a nanotube/Teflon composite (91). The electrocatalytic
Herzog and Arrigan explored various self-assembled mono-
properties of the material toward NADH and hydrogen peroxide
layers, capped with sulfonate and carboxylic groups, on gold
were not impaired, which was used for biosensing of ethanol and
electrodes to reduce surfactant inhibition on the detection of
glucose at low potentials by incorporating suitable enzymes into
copper ions by anodic stripping underpotential deposition (100).
the electrode matrix. In a similar approach, Valentini et al.
While common surfactants had no effect on the calibration curves,
compared carbon nanotube pastes, obtained by oxidative purifica-
detection limits for copper were only in the micromolar range.
tion of such nanotubes followed by mixing with mineral oil, to
Building on earlier efforts by others, bismuth film electrodes were
traditional carbon paste and found significantly improved elec-
used by the group of Smyth as a mercury-free material for the
troanalytical properties for the oxidative detection of dopamine
simultaneous adsorptive stripping analysis of cobalt and nickel
ions, although detection limits were found to be higher than with
Wang’s group developed an electrochemical sensor for the
continuous monitoring of the explosive 2,4,6-trinitrotoluene (TNT)
Microelectrodes. Microelectrodes possess numerous advan-
in untreated marine environments with 25 ppb detection limits
tages that make them highly attractive in chemical sensor research
(93). The sensor operated by square wave voltammetry at a
and scanning electrochemical microscopy. The groups of White
carbon-fiber electrode, and oxygen background was corrected for
and Amatore developed nanometer-sized (2-150 nm) platinum
by a computerized baseline subtraction.
electrodes by electrophoretic coating of etched Pt wires with poly-
The group of Meyerhoff continued research on nitric oxide-
(acrylic acid) (102). Fundamental electrochemical studies on such
releasing materials for improved in vivo biocompatibility by
electrodes showed that as few as 7000 molecules can be detected.
designing an intravascular amperometric oxygen sensor contain-
In another approach, Abbou et al. fabricated submicrometer-sized
ing an NO-releasing silicone rubber coating (94). The NO-
electrodes by melting the tip of Au microwires with an electric
releasing diazeniumdiolated secondary amines were covalently
arc followed by insulation with electrophoretic paint, which was
attached to the silicone rubber. In vivo studies of the catheters
electrochemically removed just at the very tip (103).
over a 16-h period showed no significant platelet adhesion or
The group of Cooper systematically studied the effects of
thrombus formation, and data from the improved oxygen sensors
microelectrode array geometries (center-to-center spacing and
correlated well with in vitro values. Robins and Schoenfisch applied
electrode size) on their voltammetric behavior in view of designing
micropatterning techniques to design aminosilane containing sol-
electroanalytical sensors (104). Loosely packed microelectrode
gel surfaces that can release NO to inhibit platelet adhesion while
arrays were found to show improved response times in a
not interfering with the underlying sensing chemistry (95). The
ferrocene-mediated enzyme-linked assay configuration.
group of Urban studied the effect of antimicrobial treatments on
In an interesting approach, Baranski applied a high-amplitude
the cytotoxicity and cytocompatibility of biosensor membranes
and high-frequency alternating voltage onto microelectrodes to
based on polyurethane, with glucose biosensors as a model system
heat the local environment for enhanced electrochemical detection
(96). While toxicity of membrane eluates could be eliminated by
(105). Apparently, superposition of this heating waveform does
washing steps, even after a chemical treatment, the rate of cell
not interfere with normal electroanalytical measurements. Such
growth on the membranes themselves depended on the type of
hot microelectrodes possess special promise for the detection of
analytes that are kinetically difficult to oxidize or reduce.
Zhang’s group developed a nitric oxide sensor with detection
Microelectrodes were used in sophisticated arrangements to
limits down to 0.3 nM by direct and selective oxidation of nitric
probe redox-active analytes in confined samples of biological
oxide by an array of microelectrodes, which was coated with layers
relevance. A very important area of research continues to be the
of the cation exchanger Nafion and a commercial nitric oxide-
study of neurotransmitters on a single-cell level. The group of
selective membrane (97). The sensor discriminated about 1000-
Ewing reported on a liposome model to understand the escape
fold against dopamine and 10 000-fold against the typical inter-
of transmitters from synapses in vivo, with an emphasis on the
different processes (diffusion vs flow) that dictate transmitter
While molecularly imprinted polymers (MIPs) are potentially
transport (106). The group of Wightman used a ∼100-pL transpar-
highly attractive materials for chemical sensing, few truly selective
ent fused-silica vial containing a Ag/AgCl reference electrode that
sensors have been developed so far. In most successful electro-
was capped from the outside electrolyte with a drop of oil to study
chemical cases, the analyte is directly electrolyzed at an electrode
single-cell uptake processes with carbon fiber microelectrodes that
coated with a MIP, which acts as a selective membrane. An
were inserted into the vial (107). In this elegant work, dopamine
example for this approach was given by Shoji et al., who developed
was injected into the vial, which was shown not be depleted by
an atrazine sensor based on a MIP composed of methacrylic acid
the continuous fast scan cyclic voltammetry detection unless a
and a cross-linker on a gold electrode (98). While the measuring
single cell was present that was designed to uptake dopamine.
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
Cyclic voltammetry was preferred over amperometry to preserve
sensors for gases other than oxygen, such as NOx, CO, H2, and
the analyte in the vial. The same authors showed that cyclic
hydrocarbons (37 refs) (120). Similarly, Opekar and Stulik
voltammograms can be deconvoluted to remove the temporal lag
reviewed the status of amperometric solid-state gas sensors with
due to adsorption and desorption of catecholamine, leading to
an emphasis on electrode and electrolyte materials used to achieve
similar effective response times as with amperometry (108). In a
adequate catalytic activity and size of the three-phase boundary
different approach, Yasukawa et al. fabricated a 100-pL cell by
between electrode, electrolyte, and gas (121).
electrochemical back-etching of a sealed gold wire (109). Single
Original Papers. While many gas sensor arrays have been
plant cells were then inserted, and cell metabolites released into
termed electronic noses in the past few years, research has thus
the vial were measured with electrochemical enzyme assays. In
far focused on the development of gas sensors and the chemo-
a more elaborate approach, picoliter-sized wells approaching the
metric analysis of the resulting data. The group of Walt has, for
size of single cells were micromachined onto silicon chips and
the first time, explored the effect of the nasal cavity flow
the exocytosis of catecholamine was monitored amperometrically
environment by constructing a simpler version of such a cavity
(110). Because of the optimized geometry of the well, a large
as a plastic model (122). While this preliminary study was done
fraction of the released catecholamine could be detected with
with fiber-optic sensors, it was found that not only the sensitivity
millisecond time resolution. Extracellular hydrogen peroxide levels
but also the selectivity of the sensor response varies drastically
of the brain of living rats were monitored by the group of Michael
as a function of position in the nasal cavity.
with amperometric microelectrodes modified with a cross-linked
The group of Zellers recently concluded that even relatively
redox polymer containing horseradish peroxidase (111). This
sophisticated nonspecific gas sensing arrays are not capable of
work shows that enzyme-modified electrodes can be reliably used,
reliably determining complex, real-world gas mixtures. As a result
thereby expanding the range of analytes that can be detected with
of this, sensing arrays capable of distinguishing up to three gases
such microelectrodes, although this goes at the expense of
in a mixture are now developed as a chemically sophisticated
temporal resolution. Microelectrodes were also explored by the
detector in portable gas chromatography devices. In a recent work,
group of Compton for the determination of hydrogen sulfide in a
they have characterized chemiresistive vapor sensor arrays on
Clark-type configuration where a membrane separates the inner
the basis of spray-coated gold-thiolate monolayer-protected
chamber from the sample (112). The observed current was found
nanoclusters for the detection of 11 different organic solvent
to be independent of the membrane used, which was explained
vapors, with 700 parts per trillion detection limits for most tested
by the reduced diffusion layer thickness associated with the
microelectrode compared to larger electrode configurations.
Dravid’s group used site-specific dip-pen nanopatterning of
MacPerson et al. imaged the diffusion of redox-active probe
precursor inks to fabricate small chemiresistive tin oxide semi-
molecules through isolated 100-nm-diameter pores of track-etched
conductor sensors sensitive to reducing or oxidizing gases (124).
membranes by combined scanning electrochemical-atomic force
An array of eight different gas sensors was realized with this
microscopy with platinum-coated AFM probes (113). This com-
technology by doping each ink with different metal ions, giving
bination of topographical and electrochemical information by a
different patterns when exposed to single gases such as chloro-
single probe represents a very attractive tool for spatially resolved
form, toluene, and acetonitrile. As often seen with such nonspecific
chemical analysis. This paper is just one of numerous examples
sensing arrays, no gas mixtures were tested.
dealing with such chemically selective microscopy techniques.
Lewis and co-workers explored the use of plasticizers for their
carbon black-polymer composites for use as vapor-sensitive
ELECTROCHEMICAL GAS SENSORS
detection arrays that are interrogated by resistance measurement
Reviews. Boegner and Doll reviewed the principles of semi-
(125). Adding different plasticizer concentrations was found to
conductor gas sensors based on the electroadsorptive effect,
alter the selectivity of the polymer as well as the response time,
where electrical fields applied on the gas-sensitive layer may alter
which may broaden the palette of available materials for gas
the adsorption characteristics of the material and hence the
sensing. Kaner’s group used polyaniline nanofibers for the
resulting sensing behavior (114). Nicolas-Debarnot and Poncin-
detection of gaseous acids or bases (hydrochloric acid and
Epaillard wrote a review on polyaniline-based gas sensors, cover-
ammonia) via changes in the resistance of such fiber assemblies
ing a 7-year period from 1995 (77 citations) (115). Dubbe reviewed
(126). Such nanofiber films are attractive because of their large
the principles of solid electrolyte gas sensors and their miniatur-
surface area compared to solid film sensors, although it appears
ization to thin-film microsensors (114 citations) (116). Lapham
to be difficult to adapt such intrinsically pH-sensitive materials to
et al. discussed the difficult task of developing reliable electro-
a much wider range of gaseous analytes.
chemical sensors based on proton conductors for the measure-
Knake and Hauser fabricated an electrochemical sensor for
ment of dissolved hydrogen gas in molten aluminum (117).
ozone gas with a 0.6 ppb detection limit (127). The device was
Knauth and Tuller gave a long historical overview of the principles
based on a Au-Nafion electrode with a sulfuric acid solution as
of solid-state ionics as they relate to a number of important
internal electrolyte solution. Major interferences such a nitrogen
applications, including gas sensing (292 refs) (118). Ramamoorthy
dioxide were eliminated by use of a chemical filter. The same
reviewed the principles and applications of oxygen sensors,
group reported on the detection of a mixture of electroactive gases
including the solid electrolyte types used for high-temperature
by using such Au-Nafion electrodes where electrolysis occurs
applications as well as dissolved oxygen based on the Clark
at a three-phase boundary (128). The accurate analysis of mixtures
electrode and optical sensor principles (72 citations) (119).
of three organic and four inorganic gases was possible in the ppm
Reinhardt et al. reviewed the development of amperometric
concentration range with multivariate calibration and partial least-
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
surfaces for the development and characterization of DNA sensors
The group of de Rooij reported on MOSFET gas sensors with
and enzyme biosensors (186 citations) (142).
a modulated operating temperature. When the temperature was
Enzyme Biosensors: Glucose. Glucose biosensors comprise
pulsed with a time constant of less than 100 ms, the kinetics of
the most extensively studied class of enzyme biosensors because
the gas reactions with the film was found to be modified (129).
of the relatively high durability of the enzyme, typically glucose
This discovery may be used to increase the recovery time after
oxidase, and the high practical relevance of glucose determina-
exposure to a gas such as hydrogen, and temperature cycling may
tions. To solve the problem of thermal instability, the group of
also be used to discriminate between different gases for multi-
Bachas used a new thermostable glucose enzyme, glucose-6-
phosphate dehydrogenase, obtained from the hyperthermophilicbacterium Aquifex aeolicus (143). The product of the enzymereaction, NADH, was electrocatalytically reoxidized by a thermo-
stable osmium complex at a graphite electrode. The amperometric
The field of electrochemical biosensors has seen significant
biosensor response showed excellent temperature stability even
growth in the past few years, with the development of enzyme
at 83 °C and forms a highly promising addition to modern glucose
biosensors and DNA detection principles leading the way. The
biosensor development. Most researchers in the field of electro-
following papers give just a sampling of the various approaches
chemical glucose biosensor development are targeting improve-
ments in selectivity by design of the underlying sensing material. Reviews. An Analytical Chemistry Perspectives article was
Electrochemical control of the entire deposition process has been
published by the group of Turner on the application of natural
a notable development. Parallel efforts by the groups of Wilson
receptors in biosensors and bioassays, with 92 references (130).
(144) and Schuhmann (145) found that glucose oxidase can be
The authors also outlined the challenges in view of a successful
electrochemically deposited by inducing a change in the local pH
commercialization of such sensors. Abel and von Woedtke
at the electrode surface, which changes enzyme solubility.
reviewed the status and challenges of in vivo enzyme-based
Wilson’s group studied the influence of added surfactant on the
glucose sensors (76 citations), emphasizing the importance of the
thickness of formed enzyme layer by this mechanism (144). Based
sensor surface on biocompatibility (131). The group of Heller
on this work, the group of Wilson reported on the electrochemi-
reviewed electrochemical sensors based on electrical wiring of
cally controlled deposition of a permselective layer of polyphenol
enzymes, including their recent developments of in vivo glucose
after such an enzyme deposition, which was additionally protected
sensors as well as immunosensors and DNA sensors (132). Stefan
by a (3-aminopropyl)trimethoxysilane membrane fabricated by
et al. reviewed the principles of enantioselective sensors by
electrochemically assisted cross-linking (146). This yielded du-
comparing different electrochemical sensing and recognition
rable glucose sensors with rapid response times, high sensitivity,
principles (52 citations) (133).
and low interference from undesired electroactive species. On the
other hand, Karyakin et al. used glucose oxidase embedded into
Talanta issue on DNA detection, Palecek reviewed
Nafion membranes from a water-organic solvent mixture in order
the electrochemistry of DNA for the detection of DNA damage
to stabilize the enzyme by a membrane-forming polyelectrolyte
and hybridization at attomole levels or lower (120 citations) (134).
(147). When the enzyme/Nafion casting solution was applied onto
Kelly wrote a review on the principles of charge migration trhough
Prussian Blue-modified glassy carbon electrodes (for improved
the DNA double helix and their importance to the design of
hydrogen peroxide response), good sensitivity toward glucose
electrochemical biosensors (135). Fojta reviewed the status of
electrochemical sensors for DNA interactions and damage from
Other authors continued work on the electrical wiring between
small molecules by use of either intrinsic electrochemical DNA
glucose oxidase and the underlying electrode for efficient electron
signals on redox electrodes or electroactive markers that interact
transfer. The group of Willner covalently attached N-6-(2-amino-
with DNA (158 refs) (136). Similarly, Takenaka reviewed elec-
ethyl)flavin adenine dinucleotide as a linker between glucose
trochemical techniques based on DNA intercalation by electro-
oxidase and a redox polymer composite polyaniline/poly(acrylic
active probe molecules, including so-called hybridization indicators
acid) (148). This direct electrical contact yielded very high
(137). Vercoutere and Akeson reviewed the development of
electron-transfer rates. The mechanism of such a glucose sensing
biosensors for DNA sequence detection as a replacement for
architecture was studied by in situ surface plasmon resonance,
established DNA microarrays, using electrochemical sensors and
and the sample glucose concentration was shown to control the
impedance techniques in nanoscale pores (51 refs) (138). Wang
steady-state concentration ratio of reduced and oxidized form of
wrote a review on nanoparticle-based electrochemical DNA
polyaniline. The group of Watanabe studied a series of pheno-
detection, including his own work in this area (18 citations) (139).
thiazine-labeled poly(ethylene oxide) linked to lysine residues on
The group of Willner reviewed the use of magnetic particles for
glucose oxidase as electrical wires for glucose sensing (149). A
the development of biosensors as well as electrochemical DNA
maximum catalytic current was observed for a linker size of 3000
and immunoassays (41 refs) (140). Mascini’s group reviewed the
Da. Palmisano et al. used a composite of tetrathiafulvalene-
fabrication and selection methods of aptamers and the use of these
tetracyanoquinodimethane crystals and overoxidized polypyrrole,
artificial nucleic acid ligands as affinity biocomponents in biosen-
giving a direct electrical connection between enzyme and under-
sors (so-called aptasensors), with 50 references (141). The group
lying platinum electrode (150). Efforts also continued in direction
of Willner also wrote a detailed review on the use of impedance
of miniaturization. Hrapovic and Luong, for example, fabricated a
spectroscopy as a tool to probe biomolecule interactions at
glucose biosensor with tip diameters estimated between 10 and
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
500 nm (151). The enzyme was entrapped by electropolymerized
found, and lactate sensors with detection limits down to 50 nM
phenol and 2-allylphenol, similar to the systems discussed above.
were constructed. Yu et al. reported on an efficient electrical wiring
Novel concepts for electrochemical glucose sensing were also
of enzymes for biosensor construction (160). A 4-nm layer of
reported. Tlili et al. used fibroblast cells grown on an optically
sulfonated polyaniline on a polycationic underlayer was covered
transparent indium tin oxide electrode (152). They found that the
with a film containing the enzyme (myoglobin or horseradish
electrochemical impedance response changed reproducibly with
peroxidase) and poly(styrenesulfonate). It was shown that 90%
the glucose concentration in the sample in the range of 0-14 mM,
or more of the protein was electrically coupled to the electrode,
with other sugars showing no interference.
giving an improved biosensor sensitivity with a 3 nM detection
Other Enzyme Biosensors. Site-directed mutagenesis was
used by Bao et al. to fabricate an amperometric histamine sensor
While carbon paste has been found to be an attractive matrix
with improved detection limits (153). For this purpose, phenyl-
for biosensor research because it can be doped with catalysts and
alanine 55 on a subunit of the enzyme methylamine dehydroge-
biomolecules, Mailley significantly improved such amperometric
nase was replaced by alanine, giving a 400-fold lower Km value in
biosensors by using a composite of carbon paste and in situ-
solution and a 3-fold lower value when immobilized into a
generated polypyrrole containing the enzyme polyphenol oxidase
polypyrrole sensing matrix. The resulting detection limits were
for catechol detection (161). The composite exhibited much
found to be 4-fold lower for sensors with the modified enzyme.
improved enzyme retention because of its effective entrapment
The group of Hall used site-specific mutations on trimethylamine
dehydrogenase to facilitate electrical wiring between enzyme and
Abad et al. introduced an immobilization technique to attach
redox mediators at the electrode (154). Two different mutants
glycosylated proteins covalently to self-assembled monolayers on
were designed and studied in detail, and the most promising
gold electrodes (162). Rather than using boronic acids, which
enzyme was successfully immobilized into an electrochemical
form reversible bonds with saccharides, the authors combined
sensor configuration where direct electrical wiring to an iron-based
such boronates with epoxy groups to achieve a very stable
redox polymer was confirmed electrochemically. In analogy to
their glucose work cited above, the group of Bachas developed
A method to determine the concentration and isomer ratio of
an improved biosensor for asparagine on the basis of a thermo-
urocanic acid, which is important in understanding the photo-
stable recombinant asparaginase (155). The enzyme was found
immunosupression in the skin, was developed by Tatsuma et al.
to be thermostable up to 85 °C in solution and was placed in front
by monitoring the inhibition of the hydrogen peroxide reduction
of an ammonium-selective electrode to fabricate a potentiometric
at a heme peptide-modified electrode (163). Since the two different
sensor with a 6 × 10-5 M detection limit for L-asparagine.
isomers show different inhibition of this response, the current
Naal et al. fabricated an amperometric sensor for the explosive
before and after UV irradiation, which transforms the trans into
2,4,6-trinitriotoluene (TNT) on the basis of the oriented im-
the cis isomer, could be used to estimate the isomer ratio of the
mobilization of a nitroreductase maltose binding protein fusion
(156). In contrast to the immobilized fusion protein, the wild-type
Mao et al. developed an enzyme-modified ring-disk carbon
nitroreductase alone lost most of its enzymatic activity when
film electrode embedded in a thin-layer radial flow cell for the
deposited onto the electrode modified with an electropolymerized
determination of trace amounts of hydrogen peroxide from brain
film. Detection limits for TNT were ∼2 µM.
microdialysate (164). While the ring electrode contained horse-
Aoki et al. continued their work on silicon-based light addres-
radish peroxidase for actual hydrogen peroxide detection, the disk
sable pH electrodes, where only the illuminated microdomain
electrode contained ascorbate oxidase to preoxidize and eliminate
gives rise to a potentiometric response, to the fabrication of an
ascorbic acid that would otherwise interfere with the on-line
enzyme-based multianalyte sensor for sucrose, maltose, and
analysis. In an alternate approach, Choi et al. explored the use of
glucose (157). Different spots on the chip were coated with
an insoluble oxidant membrane placed in front of an enzyme
appropriate thermophilic enzymes for improved durability and
containing film to remove interfering oxidizable species (165).
illuminated with light-emitting diodes. Chemometric analysis of
Creatinine and glucose biosensors were used as model systems,
the results was explored for better accuracy.
and the best oxidant was determined to be PbO2.
Numerous papers continued to use various polyelectrolytes
Rather than using enzymes as biocatalysts to detect their
(polymers and clays) to stabilize enzymes in biosensor configura-
substrates, Neufeld et al. electrochemically determined the
tions. For example, Kanungo et al. entrapped enzymes into poly-
enzymes released from lysed bacteria as a method to quantify
(styrenesulfonate)-polyaniline composites that were synthesized
and identify bacteria (166). In this example, a bacteriophage
within the pores of track-etched polycarbonate membranes, which
specific for Escherichia coli was used to release the bacterial cell
resulted in immobilizing the enzymes during polymerization (158).
content into solution. Amperometric detection of the marker
Compared to classical polyaniline-based biosensors, an increase
enzyme activity (a galactosidase) gave detection limits as low as
in linear response range and a decreased response time was
1 colony-forming unit/100 mL sample.
observed. A microtubule sensor array was constructed on the basis
Sun and Jin determined zeptomole quantities of enzymes from
of this principle for the simultaneous measurement of glucose,
individual human erythrocytes by electrokinetically injecting the
urea, and triglyceride in the same sample. In another example,
sample into a capillary where the sample was electromigrated to
Wei et al. used the polycationic biopolymer chitosan to form thin
a region of higher temperature to initiate enzyme reaction (167).
biopolymer films containing the polyanionic enzyme lactate
The electroactive product NADH of the model enzyme glucose-
oxidase (159). A much improved stability of the enzyme was
6-phosphate dehydrogenase used here was then monitored at a
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
carbon fiber disk bundle electrode. This is one of many examples
chemical results were compared to surface roughness experiments
of a hyphenated technique where electrochemistry is used to
using atomic force microscopy. Zayats et al. used impedance
inject, separate, and detect the analyte.
measurements on ion-sensitive field-effect transistor devices to
Immunosensors. The principles of electrochemical immuno-
determine the film thicknesses of the biomaterial, with good
sensors are now well established, and current developments go
correlations to surface plasmon resonance measurements on the
mainly in the direction of miniaturization and the fabrication of
same system (175). This device is mainly useful for the detection
array systems in the form of biosensor chips and the exploration
of large analytes such as antibodies or the toxin cholera, for which
of alternate interrogation principles. The group of Fritsch reported
on an immunoassay in a microcavity format containing a recessed
Oligonucleotides: Direct Detection. Numerous label-free
microdisk with covalently attached antibody and a nanoband gold
detection methods have been explored in the past few years. De
electrode for voltammetric detection of the enzyme label reaction
los Santos-Alvarez electrochemically oxidized the adenine bases
product, p-aminophenol, with 56-zmol detection limits for the
of adsorbed oligonucleotides on pyrolytic graphite electrodes
detection of IgG (168). In another example, Kojima et al.
(176). They found that the reaction products were electroactive
developed an electrochemical protein chip with an array of 36
and strongly adsorbed onto the electrodes, which could be used
platinum electrodes, in addition to thin-film silver/silver chloride
to detect specific DNA sequences and synthetic homopolynucle-
electrodes and auxiliary electrodes, integrated on a glass substrate
otides. In a similar approach, Jelen et al. treated DNA with a strong
(169). Immobilization was achieved by plasma polymerization of
acid to release its purine bases that were then detected by cathodic
a siloxane structure that showed no detectable nonspecific
stripping voltammetry on a copper amalgam or hanging mercury
adsorption, and independent enzyme labeled sandwich immu-
drop electrode with subnanomolar detection limits (177). Ozkan
noassays were successfully performed at different sites on the
et al. used the direct electrochemical oxidation of the guanine
bases by differential pulse voltammetry at a carbon paste electrode
The group of Smyth developed a competitive electrochemical
to monitor the hybridization of DNA (178). Since peak currents
enzyme-labeled immunoassay for sequential analyses of atrazine
for this assay were different for an allele-specific mutation, a label-
without any washing or regeneration steps (170). This was
free yes/no system for the desired mutation was developed. The
achieved by allowing the redox centers of the horseradish
group of Mascini also used the electrochemical response of the
peroxidase enzyme label to couple directly to the conducting
guanine bases in the target DNA in a label-free assay but
polymer substrate. Atrazine was detected down to 0.1 ppb
substituted all guanine bases in the immobilized probe DNA by
inosine (179). This assay was developed in view of the detection
Grant et al. improved on an interesting label-free and reversible
of PCR samples of 244-base pair fragments related to the
electrochemical immunosensor principle originally reported by
apolipoprotein E in just 10 min. Palecek’s group significantly
Sadik and Wallace (171). The antibodies (against bovine serum
reduced the problem of nonspecific adsorption of undesired
albumin and digoxin) were embedded into conducting polypyrrole
nucleotides at the electrode surface by physically separating the
films and interrogated by pulsed amperometry. The chrono-
recognition and detection surfaces (180). Hybridization was
amperometric responses were reversible in quiescent solutions
achieved at paramagnetic beads, followed by acid treatment that
and showed a linear measuring range between 0 and 50 ppm.
released adenine into solution that was detected at a mercury
Dai et al. proposed the use of a pseudoreagentless ampero-
electrode. This sensitive label-free method was demonstrated with
metric immunosensor based on the direct electrochemistry of
numerous types of oligonucleotides, and some possibilities to
horseradish peroxidase (172). This enzyme was labeled to the
further increase the sensitivity through the use of catalytic
antibody for the target antigen (carcinoma antigen-125), which
schemes were discussed. See also Janata’s work above for yet
were both deposited onto the sensor platform before measure-
another example of a label-free DNA detection method (67).
ment. When increasing concentrations of antigen were present
Oligonucleotides: Intercalator Detection. The electro-
in the sample, the current from the enzyme was found to decrease
chemical detection of DNA via redox-active or electrocatalytic
because of the competition between antigen present in the sample
intercalators is an attractive approach to oligonucleotide hybridiza-
and immobilized on the electrode surface, yielding an apparently
tion measurements because the target DNA does not need to be
chemically modified. For example, Maruyama et al. developed an
Capacitance and impedance techniques are increasingly being
osmium(II) complex containing amine electron-donating groups
used to probe immunoreactions at electrode surfaces, somewhat
that showed a high binding affinity (3 × 107 M-1) to double
in analogy to surface plasmon resonance. Of course, as a label-
stranded DNA and a low half-wave potential (181). When probe
free technique, they are potentially very versatile but more prone
DNA was immobilized onto a gold electrode, detection limits for
to effects from nonspecific adsorption than established voltam-
the electrochemical determination of target DNA was found to
metric techniques using an enzyme label, for example. The group
be 0.1 ng L-1 with a wide linear range. In a related approach,
of Sadik used differential impedance spectroscopy to monitor the
albeit not with a classical intercalator as reporter molecule,
kinetics and surface loading of protein immobilization and
Masarik et al. proposed the adsorptive transfer stripping square
antibody-antigen reactions as a fundamental technique to un-
wave voltammetric detection of streptavidin and avidin to quantify
derstand surface deposition mechanisms and surface reactivity
DNA hybridizations of biotinylated oligonucleotides (182). Detec-
(173). Corry et al. also probed antibody-antigen binding events
tion limits were found to be as low as 6 pM for denatured
at gold-coated quartz crystals and indium-doped tin oxide films
streptavidin. Homberg and Thorp performed an electrochemical
by electrochemical impedance spectroscopy (174). The electro-
study and digital simulation to quantify the binding and rate
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
constants for the reaction of DNA with two different intercalators
electrode. Upon hybridization with the target 121-nucleotide
used simultaneously, one acting as an electrocatalyst for guanine
sequence, a secondary DNA probe tagged to alkaline phosphatase
oxidation, giving higher currents with higher double-stranded
was hybridized. The enzyme generated aminophenyl phosphatase,
DNA concentrations, and the other used as redox probe, giving
from its added substrate p-aminophenol, which was detected
lower currents in the presence of DNA because of decreased mass
electrochemically at the gold electrode. Detection limits were
transport (183). Wong and Gooding explored a mixed monolayer
found to be ∼150 nM, with good selectivity. Kim et al. used the
on gold containing single-stranded DNA and incubated with the
same enzyme reaction in a related DNA assay, but by using an
redox-active intercalator 2,6-disulfonic acid anthraquinone for DNA
aminated dendrimer containing ferrocenyl groups as electrocata-
detection (184). Only when complementary DNA was allowed to
lyst between the self-assembled monolayer and the DNA probe
interact with the monolayer were voltammetric peaks for the
to increase sensitivity (192).
oxidation and reduction of the intercalator observed, indicating
The group of Heller used a carbon electrode chemically
that the double-stranded DNA was needed for electron transfer.
modified with a redox polymer and electrodeposited avidin to
Binding to DNA with mismatched base pairs gave reduced signals.
construct a sandwich assay, with probe DNA or RNA binding to
Yang et al. used the polymerase chain reaction to amplify the
the target, which in turn binds to an enzyme-labeled oligonucle-
desired DNA with 7-deaza analogues of guanine and adenine in
otide delivered to the sample (193). The electrode was made
order to obtain a larger electrochemical oxidation current in the
specific by conjugating biotinylated probe RNA or DNA to the
presence of a ruthenium(II) bipyridine as electrocatalyst (185).
deposited avidin. Upon cohybridization with the target oligonucle-
Fahlman and Sen proposed molecular design strategies in order
otide and a horseradish peroxidase-tagged oligonucleotide, a
to use the change in electron-transfer properties of double-
sandwich was formed that was interrogated electrochemically by
stranded DNA as an aptamer for the detection of intercalators,
measuring the hydrogen peroxide reduction current. The electrical
not the other way around (186). However, the selectivity of such
wiring of the enzyme with the redox polymer, which this group
a sensor was not yet characterized and is perhaps quite limited.
has already successfully used for glucose sensor development, is
Mugweru and Rusling developed a self-contained probe for
one of the key features of this electrochemical assay that takes
damaged DNA with a catalytic film containing the DNA inter-
∼30 min to complete. Subsequent work of the same group used
calator ruthenium-bipyridine and square wave voltammetric
a microelectrode configuration for increased mass transport and
detection (187). When double-stranded DNA was subjected to the
achieved a 100-fold improvement in detection limit down to ∼20
suspected carcinogen styrene oxide, the catalytic current was
pM levels (194). Williams et al. developed a related method for
found to increase linearly with time. The mechanism of this assay
rapid DNA screening, using a very simple modified streptavidin
was explained with the catalyst having improved access to the
carbon-polymer composite electrode that can be renewed by
oxidizable bases of the damaged and partly unwound DNA,
polishing between measurements (195). In this approach, target
thereby increasing the current compared to undamaged DNA. In
and probe DNA and a horseradish peroxidase enzyme label bound
a related approach, Zhou et al. screened for DNA damage by
to a suitable antigen are all added to the sample at the same time,
forming a multilayer thin film containing the double-stranded DNA
eliminating separate binding and washing steps.
of interest and myoglobin or cytochrome P450 (188). Upon
Willner’s group utilized an enzyme label for DNA detection
activation with acid, sample styrene was converted to the carci-
that produces an insoluble reaction product (196). The readout
nogenic styrene oxide in situ by the enzyme and the intercalators
was accomplished by impedance spectroscopy (and by a quartz
ruthenium(II) and cobalt(II) bipyridine were used to electro-
crystal microbalance), and DNA detection limits were found to
chemically distinguish between intact and damaged DNA. Kelley’sgroup used the electrocatalytically enhanced voltammetric ruthe-
nium(III) hexamine response to monitor DNA hybridization at a
Oligonucleotides: Nanoparticles and Quantum Dots.
gold electrode (189). Since the ruthenium complex interacts
Nanoparticle labels for oligonucleotides are known to be very
electrostatically with DNA, it leads to a larger current when
attractive in spectroscopic readout methods and share unique
hybridized DNA is present. A single base pair mismatch could
properties that are very useful for electrochemical detection as
be identified by following the voltammetric response as a function
well. In many cases, metal nanoparticles can be oxidized to form
of hybridization time. Yamashita et al. found in a detailed study
metal ions that are conveniently determined electrochemically. A
that single base pair mismatches in DNA assays involving a 20-
recent example for this approach was described by Oxsoz et al.,
mer probe can be electrochemically identified by using the
who monitored the direct oxidation current of gold nanoparticle
intercalator ferrocenylnaphthalene diimide (190). Quartz crystal
tags upon hybridization of tagged target DNA and probe DNA
microbalance and MALDI-TOFLMS studies confirmed that the
covalently attached onto a graphite electrode (197). In another
number of binding intercalator molecules decreased with increas-
approach, the group of Wang used gold nanoparticles coated with
ferrocenylhexanethiol and streptavidin (the latter for attachment
Oligonucleotides: Enzyme Amplified. Enzyme-amplified
of the biotinylated DNA probe) (198). Upon forming of a DNA
electrochemical oligonucleotide assays have been developed in
sandwich, the ferrocene groups were detected electrochemically
analogy to earlier enzyme-labeled immunoassays. Aguilar and
with a linear measuring range for DNA between 7 and 150 pM.
Fritsch introduced such an adaptation to a classical sandwich assay
The main advantage of this method lies in its experimental
to detect Cryptosporidium parvum in water samples (191). The
simplicity since no enzyme or enzyme substrate is needed and
probe DNA was attached via its 5′-amine terminus to a self-
amplification is achieved by the large number of ferrocene groups
assembled monolayer of mercaptoundecanoic acid on a gold
Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
Recently, such nanoparticle tags have been combined with
been explored. Affinity sensors using DNA or immunological
magnetic particles for the purpose of additional preconcentration
recognition units are perhaps not classical sensors because they
(199). Here, magnetic particles with probe DNA were used to
lack in many cases reversibility. Yet, the number of electrochemi-
capture target DNA, which in turn was allowed to hybridize with
cal detection schemes for measuring these extremely important
a secondary probe DNA tagged to a given metal nanoparticle. After
analytes is very inspiring. They range from direct label-free
hybridization, the ensembles were preconcentrated magnetically
detection principles or intercalator-based techniques, the use of
at an electrode and the nanoparticles were oxidized chemically
enzyme labels that form electroactive or insoluble products, to
and detected by anodic stripping voltammetry. The authors
the application of quantum dots and magnetic particles as labels.
demonstrated simultaneous DNA assays with 0.3 nM detection
In many of these detection principles, extremely low levels of
limits by introducing up to three different nanoparticle tags (ZnS,
detection with excellent selectivity and the capability of detecting
CdS, and PbS) that could easily be electrochemically resolved.
single base pair mismatches has been demonstrated.
The same authors also introduced polystyrene beads containing
Clearly, the area of electrochemical sensor research is very
defined amounts of various nanoparticles as electrochemical
active and fruitful. It must be emphasized that many of the
encoded tags in complete analogy to fluorescent polystyrene tags
challenges that remain in some cases, especially in the area of
used in flow cytometry or random fiber-optic arrays (200). The
selectivity, may be overcome by their integration into more
electrochemical signatures were found to correlate well with the
complex analytical systems that combine online sampling and
original nanoparticle loading concentrations.
separation steps. However, in the cases where direct detection inunmodified samples is possible, the high analysis speed and the
capability of detecting extremely small volumes without signifi-
The topic of electrochemical sensors is already quite vast and
cantly perturbing the sample remain highly attractive character-
continues to grow and broaden. The field of potentiometric
sensors, as a mature technology, has experienced importantchange in the past few years. The principal developments in this
area focus on reducing the detection limit to true trace levels,
This author gratefully acknowledges the National Institutes of
Health and the Petroleum Research Fund (administered by the
down to the low parts per trillion concentration range, and there
American Chemical Society) for supporting his research on electro-
are important advances in the areas of materials and active
components design. Importantly, potentiometry and the field ofion-transfer voltammetry start to approach each other to the extent
Eric Bakker is currently an Alumni Professor in the Department of
that the design of instrumentally controlled ion-selective electrodes
Chemistry at Auburn University. After undergraduate and graduate studiesof chemistry and analytical chemistry with the late Wilhelm Simon at the
now becomes possible. Voltammetric sensor development focuses
Swiss Federal Institute of Technology in Zurich, Switzerland, he pursued
on further miniaturization, the reduction of the addressable sample
postdoctoral studies at the University of Michigan. He joined the facultyat Auburn University in 1995 as an Assistant Professor and was promoted
volume, and the application to difficult in vivo and environmental
to Associate Professor in 1998 and to full professor in 2003. His researchinterests include fundamental and applied aspects of potentiometric,
sensing situations. Moreover, numerous materials characteristics
voltammetric, and optical sensors based on molecular recognition and
are being improved to achieve improved selectivity as well as a
extraction principles. He has published about 120 papers in this field.
larger potential window in aqueous samples. Electrochemical gassensors 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
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Anal. Chem. 2002, 74, 420A.
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or, at the most ternary mixtures, but normally fail at analyzing
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Electrochemical biosensor concepts are a vast area of research
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Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
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Afghan Political Parties: a short outline By René Teijgeler Summary Political parties are controversial in Afghanistan. Associated with recent conflict and ethnic or military factions, they are not considered a potentially positive force by the public or the Afghan Government. Strong ties to tribal, regional, religious, or ethnic identities, the lack of class awareness, and the very s
CENTRAL RECRUITMENT AND PROMOTION DEPARTMENT, CORPORATE CENTRE, MUMBAI. ADVERTISEMENT NO. CRPD/C RECRUITMENT IN THE CLERICAL CADRE POSTS OF PHARMACISTS, CONTROL ROOM OPERATORS AND ARMOURERS 1) Online registration of Application From : 08/11/2013 2) Last date for online Registration of application : 22/11/2013 3) Payment of fees - online : 08/11/2013 - 22/11/2013 4) Payment