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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- 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 [9]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, [9]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.
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
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- BIOSENSORS
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 CONCLUSIONS
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 ACKNOWLEDGMENT
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 LITERATURE CITED
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