Anal. Chem. 2004, 76, 3285-3298
Electrochemical Sensors Eric Bakker
Department of Chemistry, Auburn University, Auburn, Alabama 36849 Review Contents Potentiometric Sensors Reviews Theory and Characterization Detection Limit Component Design Membrane Materials Reference Electrodes Voltammetric Sensors Reviews Interrogation Principles Sensors Based on Electrochemically Driven Extraction Electrode and Coating Materials Microelectrodes Electrochemical Gas Sensors Reviews Original Papers Biosensors Reviews Enzyme Biosensors: Glucose Other Enzyme Biosensors Immunosensors Oligonucleotides: Direct Detection Oligonucleotides: Intercalator Detection Oligonucleotides: Enzyme Amplified Oligonucleotides: Nanoparticles and Quantum Dots Conclusions Literature Cited
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This review on electrochemical sensors covers the full calendar years of 2002 and 2003. This review is concerned with the status of the actual development of electrochemical sensing principles and covers potentiometric sensors, reference electrodes, voltammetric sensors, electrochemical biosensors (enzyme electrodes and affinity-based sensing principles), and electrochemical gas sensors. Note that some gas sensing principles are also covered in the topic of voltammetric sensors. The area of electrochemical sensors continues to broaden and blend with many other topics, including some for which other fundamental reviews are being written. For instance, electrochemical principles for the detection of analytes are highly relevant in microfluidics and the broader field of separation science for the purpose of injection, pumping, valving, and detection. The moving of droplets by the electrowetting effect is based on electrochemical principles. Contactless conductivity detectors are electrochemical detectors, even though they are not sensors in the classical sense because they lack selectivity. Scanning electrochemical microscopy and chemically selective scanning tunneling microscopy are really spatially resolved electrochemical 10.1021/ac049580z CCC: $27.50 Published on Web 05/01/2004
© 2004 American Chemical Society
sensors, even though they are classified as microscopic techniques. 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 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. This author therefore apologizes to anybody who feels that some key papers have been left out. POTENTIOMETRIC SENSORS Reviews. The group of Bachas published a broad review on ionophore-based potentiometric and optical sensors, with 149 references, aimed at a more general analytical readership, emphasizing mechanistic principles, recognition elements, and most important applications (1). Umezawa et al. wrote two comprehensive updates of their reference work on selectivity coefficients of ion-selective electrodes (ISEs) that cover papers from 1988 to 1998 (2, 3). Besides actual numerical selectivity coefficients, the reviews also report on the methodology of determination, response slopes, ionophore structures, and chemical compositions. The review on inorganic anions comprises 72 pages (references are listed separately on each page) (2), while the one on organic ions contains 105 pages (3). Macca wrote a critical review on the inconsistencies of published selectivity determinations performed in 2000 and 2001, with 68 citations (4). He suggested that much of the literature data is still of limited significance to other researchers, despite clearer guidance given in the past few years. An Analytical Chemistry A-page article was written on the principles and possibilities of low detection limit potentiometric sensors (30 refs) (5). Bobacka et al. reviewed the application of conducting polymers to potentiometric sensors, with Analytical Chemistry, Vol. 76, No. 12, June 15, 2004 3285
230 references (6). Such polymers are primarily used as inner reference elements, but have been explored as ion-selective membrane materials as well. Theory and Characterization. A detailed extension of earlier work on the theory of so-called non-Nernstian equilibrium responses of ionophore-based ISEs was presented by Amemiya et al. (7) Non-Nernstian slopes based on zero current sample concentration polarizations, which are important for low detection limit ISEs, were not discussed here. As long as the ionophore complexes primary and secondary ions independently, the model shows how the observed electrode slope must change as a function of the charges of ions and ionophore involved. This elegant work has important implications for future ionophore and membrane design. The determination of stability constants of ion-ionophore complexes directly in ion-selective membranes has been a challenge for many years. Among the methods recently introduced, the so-called sandwich membrane method, which utilizes a concentration polarized membrane made up of two segments of known composition, is one of the most powerful. Mikhelson et al. have made their early work, published in Russian, more accessible and discussed the method in depth, focusing on their titration technique, the determination of complex stoichiometries, and estimation of possible biases such as ion pair formation and diffusion potential (8). Among other papers, this method was utilized by Ceresa et al. to characterize the binding properties of a highly halide selective ionophore, [9]mercuracarborand-3 (9), which had been introduced earlier by Bachas and Hawthorne. The complex formation constants observed for this ionophore corresponded quantitatively to extraction constants determined with optical sensors based on the same ionophore, showing once more that diffusion potentials are generally not significant with ion-selective membranes. The protocols for determining complex formation constants by this method were extended to the quantification of membrane acid dissociation constants of H+selective ionophores and formation constants for electrically charged anion ionophores (10). The sandwich method was also found to be useful for the quantification of electrolyte coextraction processes (11). When one membrane segment contained a cation exchanger and the other an anion exchanger, the observed potential of the combined sandwich could be related to the coextraction constant of the respective electrolyte, which in turn was used to successfully predict the upper detection limit of ionophore-based ISEs. While the sandwich membrane method mentioned above has been used earlier for the determination of ionic impurities in ISE membranes, Gyurcsanyi and Lindner have used an optical method to do the same, where the level of protonation of a dilute electrically neutral lipophilic pH indicator in the membrane is quantified (12). This method works when ion-exchange and electrolyte coextraction processes are experimentally excluded. De Marco and co-workers continued to apply an array of surface characterization techniques to the understanding of ionselective electrodes. In one example, the fouling of mercury(II) chalcogenide electrodes upon prolonged contact in saline solution was studied by XPS, SIMS, RDE-EIS, and SR-GIXRD, and it was found that fouling is linked to the poisoning by silver salts (13). 3286
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Detection Limit. The discovery that the detection limit of ionophore-based ISEs can be lowered to trace levels has initiated interesting research by a number of groups. A steady-state model was developed to predict the lower detection limit of ionophorebased ISEs (14). Importantly, this model allows the experimentalist to calculate optimal inner solution compositions so that rational design becomes possible. Experiments with silver-selective membranes correlated well with predictions when an ion-exchange resin was used to buffer the inner solution, to avoid undesired/ unknown extraction processes from the inner membrane side. The detection limit of anion-selective electrodes was also evaluated (15). It was found that the inner solution plays a minor role with ionophore-free membranes and that the detection limit of an iodide-selective electrodes based on the [9]mercuracarborand-9 ionophore could be lowered to nanomolar levels. In all cases, hydroxide was found to be the primary sample interference that dictates the detection limit via counterdiffusion fluxes. Michalska et al. have published papers on lowering the detection limits of ISE with a conducting polymer solid contact (16). The earlier water layer theory of Pretsch was not considered here. Rather, it was argued that the self-discharge of the conducting polymer caused the very high detection limit ordinarily observed with such systems and that an anodic coulometric control could be used to reduce the detection limit by ∼3 orders of magnitude to submicromolar levels. The observed detection limits are still much higher than that observed with optimized liquid contacts, however. The group of Pretsch found that the use of microspheres normally used in HPLC, placed on the sample side of ISE membranes, completely eliminated undesired super-Nernstian response slopes to give subnanomolar detection limits for calcium (17). Pretsch also used a rotating electrode configuration, with the membrane placed off-center of the rotating axis, to yield detection limits in the picomolar range and rapid response times (18). In other work, an electrode rotator was used as a diagnostic tool to evaluate the level of optimization of the ISE in terms of its detection limit (19). De Marco’s group studied the effect of diffusion fluxes on the detection limit of the commercially available jalpaite copper ionselective electrode (20). Indeed, rotating disk electrode experiments revealed that the detection limit could be lowered to about nanomolar levels. The detection limit could be further decreased by using modified membrane compositions to further minimize copper dissolution, using an excess of sodium sulfide. Component Design. Polymer membrane-based ISEs require ion-exchanger properties, and historically, tetraphenylborate derivatives have been incorporated for cation-selective electrodes. Unfortunately, they have limited chemical stability and lipophilicity and are very difficult to covalently anchor onto the polymeric backbone. In response to this problem, perhalogenated closododecacarboranes have been introduced as alternative compounds in ISE membranes, with characteristics that equal or surpass that of the best available tetraphenylborates (21). In subsequent work, this class of compounds was covalently attached onto a methacrylic copolymer, and the ion selectivity of the membrane was perfectly retained (22). On the basis of this ion exchanger, a calcium-selective membrane based on a plasticizer-free polymer, with chemically attached calcium ionophore and cation exchanger, was successfully fabricated for the first time (22).
It is known that calcium-selective ionophores form very stable complexes in the membrane. For this reason, the resulting membranes are often subject to electrolyte coextraction at elevated concentrations, which leads to anion interference. The group of Nam has introduced a new tweezer-type ionophore with much lower complex formation constants (23). While the resulting selectivity is somewhat inferior to that of the best available calcium ionophores, anion interference was completely eliminated, even in calcium perchlorate solutions up to 0.1 M. Similarly, a new polymerizable derivative of ETH 129 was shown to form weaker complexes than its parent molecule ETH 129 (24). Membranes containing the covalent attached calcium ionophore showed somewhat inferior selectivity as well, but a reduced anion interference. Importantly, the covalent anchoring of this ionophore resulted in Nernstian response slopes, even with a calcium-free inner solution that would otherwise induce a strong inward ion flux. This was attributed to the reduced calcium mobility in the membrane (24). Different molecular design strategies were used by Sasaki et al. for the recognition of the ammonium ion (25). A tripodal preorganization to reject the spherical potassium was successful, but the resulting membrane suffered from calcium interference. The use of crown ethers containing bulky decalino blocking units gave the best results, with membranes that have better ammonium selectivity than any system reported to date. Uranyl salophenes have been established as very promising phosphate ionophores but have traditionally suffered from chemical decomposition in contact with phosphate solutions. Wojciechowski et al. have used a modified inner solution, in analogy to work on low detection limit ISEs, that effectively buffers both phosphate and uranyl ions (26). The resulting ion flux in the direction of the inner solution keeps the membrane mostly phosphate-free, thereby increasing the lifetime of such membranes to over two months. Bobacka and co-workers explored the recognition of aromatic cations such as N-methylpyridinium based on π-coordinating carriers that were either electrically charged or neutral, in membranes containing different plasticizers and backside contacted with a polythiophene-type solid contact (27). Only electrically charged carriers were found to show significant changes in selectivity. The group of Nam has assessed so-called tweezer-type carbonate ionophores to the measurement of carbon dioxide in seawater (28). With one of the ionophores, a very high carbonate selectivity over chloride (log Kpot, -6) and other minor ions was observed, sufficient for direct determination in seawater without sample pretreatment. A comparison of seawater analyses to reference methods was successfully performed. Malinowska et al. studied zirconium(IV)porphyrins as electrically charged ionophores for fluoride (29), which was preferred over all other tested ions, including the lipophilic perchlorate. A super-Nernstian response slope was observed because of hydroxybridged porphyrin dimer formation as confirmed by UV/visible spectroscopy. Membrane Materials. Kimura et al. have continued to study their thermotropic liquid crystals as ion-selective membranes doped with crown ether ionophores (30). They found that an ordered arrangement of the ionophore (confirmed by polarized
IR spectroscopy and X-ray diffraction) went along with significantly enhanced ion selectivity, which they explained with cooperative interaction of adjacent crown ethers. Polymeric membranes modified with Zeolite particles were used by the group of Walcarius for the preparation of ammoniumselective ISFETs (31). While a very high Zeolite content was needed (43 wt %) and the observed electrodes slopes were severely sub-Nernstian, a low detection limit of 10 nM was reported. An anodically electrodeposited iridium oxide pH microelectrode was fabricated and characterized by Bezbaruah and Zhang (32). While response times were fast and the pH measuring range was large, interference from redox-active compounds was significant. Yamamoto et al. fabricated a tungsten nanoelectrode (with 400-800-nm tip diameters) by etching a tungsten wire followed by electrooxidation to tungsten oxide (33). The electrode, which gave a measuring range between pH 2 and 12, was applied to extracellular pH measurements on endothelial cells. The group of Bachas used an ISE membrane covered with a polymer containing phosphorylcholine functionalities, which mimic the polar groups on cell surfaces (34). Decreased adhesion and activation of platelets was demonstrated by immunostaining, and ISE sensing characteristics were not affected by the coating. See also Voltammetric Sensors for other approaches to biocompatibility. A novel potentiometric measuring principle for the detection of saccharides was developed on the basis of poly(aniline boronic acid) (35). The complexation resulted in a change of the effective pKa of the polymer, which gave rise to a change in the observed potential. Additives such as Nafion or sodium fluoride also had a marked effect on sensor sensitivity. van der Wal et al. introduced a technique for the simple covalent attachment of poly(vinyl chloride) membranes to solid substrates such as glass and other oxide surfaces (36). Membranes attached via a silane coupling agent containing an amine group and a heating step showed excellent adhesion properties and retained their sensing characteristics relative to membranes cast without linking agent. This work potentially solves a longstanding problem in the development of ion-selective field effect transistors, without the need for a new polymeric material. REFERENCE ELECTRODES Very few works reported on new reference electrode principles. Langmaier and Samec explored freshly polished copper wires directly inserted into an electrolyte solution of the solvent o-nitrophenyl octyl ether as a junction-free inner reference electrode in ion-transfer voltammetry (see topic below) (37). They reported impressive stabilities on the order of 2 mV over 300 h, but the exact mechanism of this behavior could not be explained. Lee and Sohn explored field effect transistor-type reference electrodes consisting of a one ISFET gate covered with a pHinsensitive polymer double layer that was used in conjunction with a pH ISFET for pH detection (38). The mechanism for its functioning remains unclear as well. VOLTAMMETRIC SENSORS Reviews. Wang reviewed the current status and future challenges of miniaturizing electroanalytical systems, their incorporation into microfluidic devices, and their application to point Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
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of care and environmental analysis as well as genetic testing, with 17 references (39). In another review, the same author stressed the usefulness of electrochemical sensors for environmental monitoring applications as an approach to a greener analytical chemistry compared to traditional instruments (35 refs) (40). The group of Wightman wrote an A-page article on their work on the development of electrochemical sensors for the neurotransmitter dopamine, with 30 citations (41). Within a special issue of TrAC, Trends in Analytical Chemistry on microelectrodes and microdialysis probes for neuroanalysis, Wightman’s group also discussed characterization and validation procedures and required selectivities of such microelectrodes when used for the measurement in the brain (22 refs) (42). The status of development of electrochemical arsenic sensors for environmental monitoring applications was reviewed by Feeney and Kounaves as part of a special Talanta issue on arsenic detection, including their own work on portable systems based on microfabricated gold arrays (35 refs) (43). The status of electrochemical sensors for occupational and environmental health applications was reviewed by Ashley with 109 citations, with an emphasis on rugged and miniature electroanalytical devices for on-site monitoring (44). Methods such as disposable screen printing technology for the fabrication of sensors for trace metal pollutants in a variety of sample matrixes was reviewed by Honeychurch and Hart (46 citations) (45). The status of electrochemical sensors for the detection of metal pollutants in coastal waters was reviewed by Achterberg and co-workers, with 17 references (46). The history and current status of electrochemical nitric oxide sensors based on modified electrodes was reviewed by Bedioui and Villeneuve (129 citations) (47) as well as Ciszweski and Milczarek (with 32 refs, in a special NO detection issue of Talanta) (48). The application of self-assembled monolayers as a bottom-up fabrication principle for the realization of electrochemical sensors for pH and inorganic and biological sensors has been reviewed by Gooding et al., with 168 citations (49). This paper includes a discussion of emerging trends such as nanotubes, dendrimers, and nanoparticles for electroanalysis. Similarly, Hernandez-Santos et al. reviewed the use of metal and semiconductor nanoparticles for use in electroanalysis (59 citations) (50), Li et al. reviewed the electrochemistry at carbon nanotube electrodes in view of sensor and assay development (51), and Sherigara et al. reviewed electrocatalytic properties of nanotubes and fullerenes in view of developing electrochemical sensors, with 230 citations (52). Swain reviewed the status and future prospects of diamond science for numerous emerging technologies including electrochemistry and electroanalysis (53). The status of the development of electrochemical sensors based on molecularly imprinted polymers was reviewed by Piletsky and Turner (45 citations) (54). Similarly, Merkoci and Alegret reviewed the use of molecularly imprinted polymers in capacitive, conductometric, voltammetric, and potentiometric sensors (28 refs) (55). The group of Guilbault summarized the status of chemometrics for electrochemical sensors, with 78 citations, by focusing on multivariate calibration, classification, pattern recognition, and signal processing (56). Heineman’s group reviewed their own work on spectroelectrochemical sensing, where electrochemistry, spectroscopy, and selective partitioning are combined into a single sensing device for improved selectivity (43 refs) (57). The group of Martin reviewed 3288
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their work on the development membranes containing pores of molecular dimensions for new separation and electrochemical sensing applications, with 45 citations (58). Interrogation Principles. The groups of Bachas and Grimes introduced a novel measuring principle to monitor electrochemical processes without the need for electrical connections (59). Here, magnetoelastic alloy films were used as the working electrode in an electrochemical cell and the mass change on the electrode (polypyrrole deposition) was monitored via magnetic monitoring of the resonance frequency. Heineman continued his work on spectroelectrochemical sensing (see also review cited above (57)), for example, by describing a sensor for the detection of iron(II) (60). Here, a Nafion film loaded with a bipyridine ligand was used to extract iron(II) and to render it strongly absorbing by complexation. Electrochemical oxidation of this complex again rendered it colorless. In another example, the stripping voltammetric detection of lead and cadmium was monitored spectroscopically at the same time, with separate wavelength regions used for each of the two metals (750 nm for lead and 400 nm for cadmium), giving added selectivity (61). Ekeroth used interfacial capacitance measurements to monitor the interaction of phosphate monolayers with calcium and magnesium ions (62). Monitoring a non-Faradaic process, as done here, appears to be more susceptible to effects unrelated to the desired molecular interaction and should be used with caution. Indeed, this approach is an alternative to others who have used voltammetric techniques with a redox marker for quantifying surface recognition events. For example, Choi et al. used a competitive adsorption of electrochemically inactive organic molecules such as glucose with ferrocene onto a self-assembled monolayer containing a thiolated cyclodextrin (63). The oxidative current for ferrocene was indeed reduced with higher sample concentrations of glucose. A critical selectivity study of this device was not performed, however. Baca et al. coupled anodic stripping voltammetry online to ICPMS to develop a hyphenated technique with high selectivity and sensitivity (64). It was found that the electrochemical preconcentration gave detection limits down to sub-ppt levels, lower than possible with conventional ICPMS, and that it could be used to eliminate matrix effects as well. The group of Martin explored the use of nanotube membranes as ligand-gated ion channel mimics (65). Ion current through the membrane could be switched on or off by adding hydrophobic ionic species to the sample that could interact with the hydrophobic pores of the membrane that would otherwise be insulating. Sensors Based on Electrochemically Driven Extraction. The electrochemically controlled extraction of ions into sensing polymers and other water-immiscible phases is an attractive approach to chemical sensing that bridges the fields of polymer membrane-based ion-selective electrodes and voltammetry at metal electrodes. Wu et al. used electrochemical control of conductive polypyrrole films to extract, preconcentrate, and desorb ionic analytes, which were subsequently analyzed by flow injection analysis and mass spectrometry (66). The method was found to work for a variety of cations and anions and appears to be versatile. Janata’s group used cyclic voltammetry to control the exchange of chloride ions between polypyrrole and the buffer to fabricate a
label-free DNA hybridization detector (67). The probe DNA was immobilized onto polypyrrole via magnesium bridging complexes, and the hybridization event caused a change in the voltammetric behavior of the film. In other work, a new measurement protocol was introduced for ion-selective membranes that lack ion-exchanger properties (68). Here, current and potential pulses were alternated to control the extraction processes of the membrane electrochemically. The resulting responses have the same look and feel as potentiometric membrane electrodes, but the selectivity and response features can be tuned and even reversed, and the reversible detection of analytes that ordinarily give irreversible sensing responses becomes possible. As an important early example of this approach, the reversible detection of the polycation protamine was demonstrated for the first time (69). In parallel work, Amemiya and coworkers used cyclic voltammetry on micropipets to demonstrate the detection of protamine (70). In a similar effort, Samec et al. used cyclic voltammetry for the electrochemical detection of the anticoagulant polyanion heparin (71). The analogy of ion-transfer voltammetry to potentiometric ionselective electrode response was also stressed by Wooster et al., who studied microparticles containing 7,7,8,8-tetracyanoquinodimethane and tetrathiafulvalene in contact with electrolyte solutions. The voltammetric waves changed as a function of the type and concentration of electrolyte and were explained by ion incorporation processes as well (72). Long and Bakker used normal pulse voltammetry on pH-sensitive polymer membranes, and an apparently Nernstian relationship between sample pH and half wave potential was also observed that correlated closely with that of corresponding ion-selective electrodes (73). Spectral imaging experiments confirmed the electrochemical results. This work forms the precursor for the pulsed galvanostatic approach mentioned above where the potential can be directly obtained from the experiment. The group of Buffle continued their work on permeation liquid membranes as selective preconcentrators for metal speciation measurements by optimizing membrane and ion channel geometry (74). In this approach, the membrane is a traditional transport membrane that works on the basis of zero current counterdiffusion fluxes for the transport and preconcentration of metal ions at the backside of the membrane for metal ion sensing. See the topic above of detection limits of ion-selective electrodes for similar mechanisms. Rahman et al. used a hybrid between ion extraction/recognition and redox electrodes by doping a thiophene-based conducting polymer, which is normally known for its electrochemically mediated extraction properties, with EDTA (75). The polymer was coated onto a glassy carbon electrode, and the metals lead(II), copper(II), and mercury(II) were deposited and subsequently reduced at the electrode, with detection limits in the subnanomolar range. Electrode and Coating Materials. Ultrasonic cavitation was used by Cordero-Rando et al. to fabricate a sol-gel graphite-based electrode material from an acidic aqueous solvent in view of developing electrochemical sensors (76). The group of Collinson used an electrodeposition process from a tetramethoxysilane sol to fabricate sol-gel silicate films that were rougher than spincoated films. Various redox molecules were electroencapsulated
during film formation (77). In other work, Khoo and Chen encapsulated methylene blue into a similar sol-gel film on glassy carbon electrodes for the electrocatalytic determination of ascorbic and uric acid (78). The simultaneous determination of these two analytes in human urine samples was demonstrated. Sol-gel technology was also used by the group of Mandler to design an molecularly imprinted polymer for iron(II) using a tris(2,2′bipyridine) complex (79). However, the achieved selectivity was not satisfactory, suggesting that the recognition and detection of organic molecules is currently a more successful approach with this technology. Domenech et al. showed that Zeolite Y containing an encapsulated triphenylpyrylium ion exhibits a markedly improved oxidative response to dopamine while inhibiting the oxidation of negatively charged interferences such as ascorbate (80). A 100fold excess of ascorbate could be tolerated in a differential pulse detection mode. The group of Walcarius used Zeolites to chemically modify carbon paste electrodes for improved electroanalytical properties (81). When Zeolite particles were used instead of the classical mineral oil binder of carbon paste, or used as an outer coating, electrodes with improved responses to copper ions were observed after an ion-exchange accumulation step. Mesoporous platinum electrodes possess an enlarged surface area that enhances their catalytic properties for chemical sensing. Consequently, Evans et al. used such materials for the enhanced detection of hydrogen peroxide (82), and Park et al. found that the normally sluggish nonenzymatic glucose response was greatly enhanced with such electrodes (83). Enhanced electrochemical sugar detection after HPLC separation was also reported by You et al. by the use of highly dispersed Ni nanoparticles in a carbon film electrode, with detection limits that were at least 1 order of magnitude lower than with traditional Ni electrodes (84). Zen et al. used copper-plated screen-printed electrodes for the selective detection of o-diphenols such as catechol and dopamine in the presence of m- and p-diphenols as well as ascorbic acid under very mild conditions (-0.05 V vs Ag/AgCl) (85). The enhanced selectivity was explained by the formation of a cyclic fivemembered complex intermediate at the copper electrode surface. Copper electrodes were also used by Paixao to determine ethanol amperometrically in beverages (86). The principle was incorporated into a flow injection analysis system and used a PTFE membrane for ethanol extraction followed by oxidation under alkaline conditions. The comparison of the data from beverage analyses with gas chromatography gave excellent agreement. Boron-doped diamond electrodes continue to be adopted for electroanalysis because of their high stability, low background current, and wide potential window. Ferro and De Battisti reported on an unprecedented 5-V potential window in aqueous solutions using fluorine-terminated boron-doped diamond electrodes (87). No electroanalytical applications were yet reported with this material. Rao et al. showed that boron-doped diamond electrodes are improved detectors for carbamate pesticides after HPLC separation, offering better electrode stability (88). If a hydrolysis step was introduced prior to separation and detection, ppb detection limits were achieved for these analytes. The group of Swain explored boron-doped diamond films as electrically transparent electrodes on quartz for spectroelectrochemical applications, obtained by microwave-assisted chemical vapor deposition Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
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(89). The optical and electrochemical properties of the films were found to be extremely stable, even in harsh environments, and found to be superior to that of traditional In-doped tin oxide thin films. In analogy to carbon paste, monocrystalline diamond paste electrodes were introduced and studied by Stefan and Bairu for the determination of iron(II) in pharmaceutical preparations (90). Wang and Musameh incorporated the electrocatalytic advantages of carbon nanotubes into a more rugged configuration by forming a nanotube/Teflon composite (91). The electrocatalytic properties of the material toward NADH and hydrogen peroxide were not impaired, which was used for biosensing of ethanol and glucose at low potentials by incorporating suitable enzymes into the electrode matrix. In a similar approach, Valentini et al. compared carbon nanotube pastes, obtained by oxidative purification of such nanotubes followed by mixing with mineral oil, to traditional carbon paste and found significantly improved electroanalytical properties for the oxidative detection of dopamine (92). Wang’s group developed an electrochemical sensor for the continuous monitoring of the explosive 2,4,6-trinitrotoluene (TNT) in untreated marine environments with 25 ppb detection limits (93). The sensor operated by square wave voltammetry at a carbon-fiber electrode, and oxygen background was corrected for by a computerized baseline subtraction. The group of Meyerhoff continued research on nitric oxidereleasing materials for improved in vivo biocompatibility by designing an intravascular amperometric oxygen sensor containing an NO-releasing silicone rubber coating (94). The NOreleasing diazeniumdiolated secondary amines were covalently attached to the silicone rubber. In vivo studies of the catheters over a 16-h period showed no significant platelet adhesion or thrombus formation, and data from the improved oxygen sensors correlated well with in vitro values. Robins and Schoenfisch applied micropatterning techniques to design aminosilane containing solgel surfaces that can release NO to inhibit platelet adhesion while not interfering with the underlying sensing chemistry (95). The group of Urban studied the effect of antimicrobial treatments on the cytotoxicity and cytocompatibility of biosensor membranes based on polyurethane, with glucose biosensors as a model system (96). While toxicity of membrane eluates could be eliminated by washing steps, even after a chemical treatment, the rate of cell growth on the membranes themselves depended on the type of treatment used. Zhang’s group developed a nitric oxide sensor with detection limits down to 0.3 nM by direct and selective oxidation of nitric oxide by an array of microelectrodes, which was coated with layers of the cation exchanger Nafion and a commercial nitric oxideselective membrane (97). The sensor discriminated about 1000fold against dopamine and 10 000-fold against the typical interferences ascorbic acid and nitrite. While molecularly imprinted polymers (MIPs) are potentially highly attractive materials for chemical sensing, few truly selective sensors have been developed so far. In most successful electrochemical cases, the analyte is directly electrolyzed at an electrode coated with a MIP, which acts as a selective membrane. An example for this approach was given by Shoji et al., who developed an atrazine sensor based on a MIP composed of methacrylic acid and a cross-linker on a gold electrode (98). While the measuring 3290
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range was only in the millimolar range, selectivity was clearly improved relative to an uncoated gold electrode. See the work of Rhaman et al. discussed above for other types of selective coatings (75). Willner’s group used imprinted membranes as coatings on field-effect transistors for the detection of triazine herbicides (99). Although sensitivities were rather small, large selectivity changes and reversals were obtained upon imprinting with various herbicide substrates, making this a promising technique. Herzog and Arrigan explored various self-assembled monolayers, capped with sulfonate and carboxylic groups, on gold electrodes to reduce surfactant inhibition on the detection of copper ions by anodic stripping underpotential deposition (100). While common surfactants had no effect on the calibration curves, detection limits for copper were only in the micromolar range. Building on earlier efforts by others, bismuth film electrodes were used by the group of Smyth as a mercury-free material for the simultaneous adsorptive stripping analysis of cobalt and nickel ions, although detection limits were found to be higher than with mercury thin films (101). Microelectrodes. Microelectrodes possess numerous advantages that make them highly attractive in chemical sensor research and scanning electrochemical microscopy. The groups of White and Amatore developed nanometer-sized (2-150 nm) platinum electrodes by electrophoretic coating of etched Pt wires with poly(acrylic acid) (102). Fundamental electrochemical studies on such electrodes showed that as few as 7000 molecules can be detected. In another approach, Abbou et al. fabricated submicrometer-sized electrodes by melting the tip of Au microwires with an electric arc followed by insulation with electrophoretic paint, which was electrochemically removed just at the very tip (103). The group of Cooper systematically studied the effects of microelectrode array geometries (center-to-center spacing and electrode size) on their voltammetric behavior in view of designing electroanalytical sensors (104). Loosely packed microelectrode arrays were found to show improved response times in a ferrocene-mediated enzyme-linked assay configuration. In an interesting approach, Baranski applied a high-amplitude and high-frequency alternating voltage onto microelectrodes to heat the local environment for enhanced electrochemical detection (105). Apparently, superposition of this heating waveform does not interfere with normal electroanalytical measurements. Such hot microelectrodes possess special promise for the detection of analytes that are kinetically difficult to oxidize or reduce. Microelectrodes were used in sophisticated arrangements to probe redox-active analytes in confined samples of biological relevance. A very important area of research continues to be the study of neurotransmitters on a single-cell level. The group of Ewing reported on a liposome model to understand the escape of transmitters from synapses in vivo, with an emphasis on the different processes (diffusion vs flow) that dictate transmitter transport (106). The group of Wightman used a ∼100-pL transparent fused-silica vial containing a Ag/AgCl reference electrode that was capped from the outside electrolyte with a drop of oil to study single-cell uptake processes with carbon fiber microelectrodes that were inserted into the vial (107). In this elegant work, dopamine was injected into the vial, which was shown not be depleted by the continuous fast scan cyclic voltammetry detection unless a single cell was present that was designed to uptake dopamine.
Cyclic voltammetry was preferred over amperometry to preserve the analyte in the vial. The same authors showed that cyclic voltammograms can be deconvoluted to remove the temporal lag due to adsorption and desorption of catecholamine, leading to similar effective response times as with amperometry (108). In a different approach, Yasukawa et al. fabricated a 100-pL cell by electrochemical back-etching of a sealed gold wire (109). Single plant cells were then inserted, and cell metabolites released into the vial were measured with electrochemical enzyme assays. In a more elaborate approach, picoliter-sized wells approaching the size of single cells were micromachined onto silicon chips and the exocytosis of catecholamine was monitored amperometrically (110). Because of the optimized geometry of the well, a large fraction of the released catecholamine could be detected with millisecond time resolution. Extracellular hydrogen peroxide levels of the brain of living rats were monitored by the group of Michael with amperometric microelectrodes modified with a cross-linked redox polymer containing horseradish peroxidase (111). This work shows that enzyme-modified electrodes can be reliably used, thereby expanding the range of analytes that can be detected with such microelectrodes, although this goes at the expense of temporal resolution. Microelectrodes were also explored by the group of Compton for the determination of hydrogen sulfide in a Clark-type configuration where a membrane separates the inner chamber from the sample (112). The observed current was found to be independent of the membrane used, which was explained by the reduced diffusion layer thickness associated with the microelectrode compared to larger electrode configurations. MacPerson et al. imaged the diffusion of redox-active probe molecules through isolated 100-nm-diameter pores of track-etched membranes by combined scanning electrochemical-atomic force microscopy with platinum-coated AFM probes (113). This combination of topographical and electrochemical information by a single probe represents a very attractive tool for spatially resolved chemical analysis. This paper is just one of numerous examples dealing with such chemically selective microscopy techniques. ELECTROCHEMICAL GAS SENSORS Reviews. Boegner and Doll reviewed the principles of semiconductor gas sensors based on the electroadsorptive effect, where electrical fields applied on the gas-sensitive layer may alter the adsorption characteristics of the material and hence the resulting sensing behavior (114). Nicolas-Debarnot and PoncinEpaillard wrote a review on polyaniline-based gas sensors, covering a 7-year period from 1995 (77 citations) (115). Dubbe reviewed the principles of solid electrolyte gas sensors and their miniaturization to thin-film microsensors (114 citations) (116). Lapham et al. discussed the difficult task of developing reliable electrochemical sensors based on proton conductors for the measurement of dissolved hydrogen gas in molten aluminum (117). Knauth and Tuller gave a long historical overview of the principles of solid-state ionics as they relate to a number of important applications, including gas sensing (292 refs) (118). Ramamoorthy reviewed the principles and applications of oxygen sensors, including the solid electrolyte types used for high-temperature applications as well as dissolved oxygen based on the Clark electrode and optical sensor principles (72 citations) (119). Reinhardt et al. reviewed the development of amperometric
sensors for gases other than oxygen, such as NOx, CO, H2, and hydrocarbons (37 refs) (120). Similarly, Opekar and Stulik reviewed the status of amperometric solid-state gas sensors with an emphasis on electrode and electrolyte materials used to achieve adequate catalytic activity and size of the three-phase boundary between electrode, electrolyte, and gas (121). Original Papers. While many gas sensor arrays have been termed electronic noses in the past few years, research has thus far focused on the development of gas sensors and the chemometric analysis of the resulting data. The group of Walt has, for the first time, explored the effect of the nasal cavity flow environment by constructing a simpler version of such a cavity as a plastic model (122). While this preliminary study was done with fiber-optic sensors, it was found that not only the sensitivity but also the selectivity of the sensor response varies drastically as a function of position in the nasal cavity. The group of Zellers recently concluded that even relatively sophisticated nonspecific gas sensing arrays are not capable of reliably determining complex, real-world gas mixtures. As a result of this, sensing arrays capable of distinguishing up to three gases in a mixture are now developed as a chemically sophisticated detector in portable gas chromatography devices. In a recent work, they have characterized chemiresistive vapor sensor arrays on the basis of spray-coated gold-thiolate monolayer-protected nanoclusters for the detection of 11 different organic solvent vapors, with 700 parts per trillion detection limits for most tested vapors (123). Dravid’s group used site-specific dip-pen nanopatterning of precursor inks to fabricate small chemiresistive tin oxide semiconductor sensors sensitive to reducing or oxidizing gases (124). An array of eight different gas sensors was realized with this technology by doping each ink with different metal ions, giving different patterns when exposed to single gases such as chloroform, toluene, and acetonitrile. As often seen with such nonspecific sensing arrays, no gas mixtures were tested. Lewis and co-workers explored the use of plasticizers for their carbon black-polymer composites for use as vapor-sensitive detection arrays that are interrogated by resistance measurement (125). Adding different plasticizer concentrations was found to alter the selectivity of the polymer as well as the response time, which may broaden the palette of available materials for gas sensing. Kaner’s group used polyaniline nanofibers for the detection of gaseous acids or bases (hydrochloric acid and ammonia) via changes in the resistance of such fiber assemblies (126). Such nanofiber films are attractive because of their large surface area compared to solid film sensors, although it appears to be difficult to adapt such intrinsically pH-sensitive materials to a much wider range of gaseous analytes. Knake and Hauser fabricated an electrochemical sensor for ozone gas with a 0.6 ppb detection limit (127). The device was based on a Au-Nafion electrode with a sulfuric acid solution as internal electrolyte solution. Major interferences such a nitrogen dioxide were eliminated by use of a chemical filter. The same group reported on the detection of a mixture of electroactive gases by using such Au-Nafion electrodes where electrolysis occurs at a three-phase boundary (128). The accurate analysis of mixtures of three organic and four inorganic gases was possible in the ppm concentration range with multivariate calibration and partial leastAnalytical Chemistry, Vol. 76, No. 12, June 15, 2004
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squares regression of the results. The group of de Rooij reported on MOSFET gas sensors with a modulated operating temperature. When the temperature was pulsed with a time constant of less than 100 ms, the kinetics of the gas reactions with the film was found to be modified (129). This discovery may be used to increase the recovery time after exposure to a gas such as hydrogen, and temperature cycling may also be used to discriminate between different gases for multianalyte detection purposes.
BIOSENSORS The field of electrochemical biosensors has seen significant growth in the past few years, with the development of enzyme biosensors and DNA detection principles leading the way. The following papers give just a sampling of the various approaches that have been explored. Reviews. An Analytical Chemistry Perspectives article was published by the group of Turner on the application of natural receptors in biosensors and bioassays, with 92 references (130). The authors also outlined the challenges in view of a successful commercialization of such sensors. Abel and von Woedtke reviewed the status and challenges of in vivo enzyme-based glucose sensors (76 citations), emphasizing the importance of the sensor surface on biocompatibility (131). The group of Heller reviewed electrochemical sensors based on electrical wiring of enzymes, including their recent developments of in vivo glucose sensors as well as immunosensors and DNA sensors (132). Stefan et al. reviewed the principles of enantioselective sensors by comparing different electrochemical sensing and recognition principles (52 citations) (133). In a special Talanta issue on DNA detection, Palecek reviewed the electrochemistry of DNA for the detection of DNA damage and hybridization at attomole levels or lower (120 citations) (134). Kelly wrote a review on the principles of charge migration trhough the DNA double helix and their importance to the design of electrochemical biosensors (135). Fojta reviewed the status of electrochemical sensors for DNA interactions and damage from small molecules by use of either intrinsic electrochemical DNA signals on redox electrodes or electroactive markers that interact with DNA (158 refs) (136). Similarly, Takenaka reviewed electrochemical techniques based on DNA intercalation by electroactive probe molecules, including so-called hybridization indicators (137). Vercoutere and Akeson reviewed the development of biosensors for DNA sequence detection as a replacement for established DNA microarrays, using electrochemical sensors and impedance techniques in nanoscale pores (51 refs) (138). Wang wrote a review on nanoparticle-based electrochemical DNA detection, including his own work in this area (18 citations) (139). The group of Willner reviewed the use of magnetic particles for the development of biosensors as well as electrochemical DNA and immunoassays (41 refs) (140). Mascini’s group reviewed the fabrication and selection methods of aptamers and the use of these artificial nucleic acid ligands as affinity biocomponents in biosensors (so-called aptasensors), with 50 references (141). The group of Willner also wrote a detailed review on the use of impedance spectroscopy as a tool to probe biomolecule interactions at 3292
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surfaces for the development and characterization of DNA sensors and enzyme biosensors (186 citations) (142). Enzyme Biosensors: Glucose. Glucose biosensors comprise the most extensively studied class of enzyme biosensors because of the relatively high durability of the enzyme, typically glucose oxidase, and the high practical relevance of glucose determinations. To solve the problem of thermal instability, the group of Bachas used a new thermostable glucose enzyme, glucose-6phosphate dehydrogenase, obtained from the hyperthermophilic bacterium Aquifex aeolicus (143). The product of the enzyme reaction, NADH, was electrocatalytically reoxidized by a thermostable osmium complex at a graphite electrode. The amperometric biosensor response showed excellent temperature stability even at 83 °C and forms a highly promising addition to modern glucose biosensor development. Most researchers in the field of electrochemical glucose biosensor development are targeting improvements in selectivity by design of the underlying sensing material. Electrochemical control of the entire deposition process has been a notable development. Parallel efforts by the groups of Wilson (144) and Schuhmann (145) found that glucose oxidase can be electrochemically deposited by inducing a change in the local pH at the electrode surface, which changes enzyme solubility. Wilson’s group studied the influence of added surfactant on the thickness of formed enzyme layer by this mechanism (144). Based on this work, the group of Wilson reported on the electrochemically controlled deposition of a permselective layer of polyphenol after such an enzyme deposition, which was additionally protected by a (3-aminopropyl)trimethoxysilane membrane fabricated by electrochemically assisted cross-linking (146). This yielded durable glucose sensors with rapid response times, high sensitivity, and low interference from undesired electroactive species. On the other hand, Karyakin et al. used glucose oxidase embedded into Nafion membranes from a water-organic solvent mixture in order to stabilize the enzyme by a membrane-forming polyelectrolyte (147). When the enzyme/Nafion casting solution was applied onto Prussian Blue-modified glassy carbon electrodes (for improved hydrogen peroxide response), good sensitivity toward glucose sensing was observed. Other authors continued work on the electrical wiring between glucose oxidase and the underlying electrode for efficient electron transfer. The group of Willner covalently attached N-6-(2-aminoethyl)flavin adenine dinucleotide as a linker between glucose oxidase and a redox polymer composite polyaniline/poly(acrylic acid) (148). This direct electrical contact yielded very high electron-transfer rates. The mechanism of such a glucose sensing architecture was studied by in situ surface plasmon resonance, and the sample glucose concentration was shown to control the steady-state concentration ratio of reduced and oxidized form of polyaniline. The group of Watanabe studied a series of phenothiazine-labeled poly(ethylene oxide) linked to lysine residues on glucose oxidase as electrical wires for glucose sensing (149). A maximum catalytic current was observed for a linker size of 3000 Da. Palmisano et al. used a composite of tetrathiafulvalenetetracyanoquinodimethane crystals and overoxidized polypyrrole, giving a direct electrical connection between enzyme and underlying platinum electrode (150). Efforts also continued in direction of miniaturization. Hrapovic and Luong, for example, fabricated a glucose biosensor with tip diameters estimated between 10 and
500 nm (151). The enzyme was entrapped by electropolymerized phenol and 2-allylphenol, similar to the systems discussed above. Novel concepts for electrochemical glucose sensing were also reported. Tlili et al. used fibroblast cells grown on an optically transparent indium tin oxide electrode (152). They found that the electrochemical impedance response changed reproducibly with the glucose concentration in the sample in the range of 0-14 mM, with other sugars showing no interference. Other Enzyme Biosensors. Site-directed mutagenesis was used by Bao et al. to fabricate an amperometric histamine sensor with improved detection limits (153). For this purpose, phenylalanine 55 on a subunit of the enzyme methylamine dehydrogenase was replaced by alanine, giving a 400-fold lower Km value in solution and a 3-fold lower value when immobilized into a polypyrrole sensing matrix. The resulting detection limits were found to be 4-fold lower for sensors with the modified enzyme. The group of Hall used site-specific mutations on trimethylamine dehydrogenase to facilitate electrical wiring between enzyme and redox mediators at the electrode (154). Two different mutants were designed and studied in detail, and the most promising enzyme was successfully immobilized into an electrochemical sensor configuration where direct electrical wiring to an iron-based redox polymer was confirmed electrochemically. In analogy to their glucose work cited above, the group of Bachas developed an improved biosensor for asparagine on the basis of a thermostable recombinant asparaginase (155). The enzyme was found to be thermostable up to 85 °C in solution and was placed in front of an ammonium-selective electrode to fabricate a potentiometric sensor with a 6 × 10-5 M detection limit for L-asparagine. Naal et al. fabricated an amperometric sensor for the explosive 2,4,6-trinitriotoluene (TNT) on the basis of the oriented immobilization of a nitroreductase maltose binding protein fusion (156). In contrast to the immobilized fusion protein, the wild-type nitroreductase alone lost most of its enzymatic activity when deposited onto the electrode modified with an electropolymerized film. Detection limits for TNT were ∼2 µM. Aoki et al. continued their work on silicon-based light addressable pH electrodes, where only the illuminated microdomain gives rise to a potentiometric response, to the fabrication of an enzyme-based multianalyte sensor for sucrose, maltose, and glucose (157). Different spots on the chip were coated with appropriate thermophilic enzymes for improved durability and illuminated with light-emitting diodes. Chemometric analysis of the results was explored for better accuracy. Numerous papers continued to use various polyelectrolytes (polymers and clays) to stabilize enzymes in biosensor configurations. For example, Kanungo et al. entrapped enzymes into poly(styrenesulfonate)-polyaniline composites that were synthesized within the pores of track-etched polycarbonate membranes, which resulted in immobilizing the enzymes during polymerization (158). Compared to classical polyaniline-based biosensors, an increase in linear response range and a decreased response time was observed. A microtubule sensor array was constructed on the basis of this principle for the simultaneous measurement of glucose, urea, and triglyceride in the same sample. In another example, Wei et al. used the polycationic biopolymer chitosan to form thin biopolymer films containing the polyanionic enzyme lactate oxidase (159). A much improved stability of the enzyme was
found, and lactate sensors with detection limits down to 50 nM were constructed. Yu et al. reported on an efficient electrical wiring of enzymes for biosensor construction (160). A 4-nm layer of sulfonated polyaniline on a polycationic underlayer was covered with a film containing the enzyme (myoglobin or horseradish peroxidase) and poly(styrenesulfonate). It was shown that 90% or more of the protein was electrically coupled to the electrode, giving an improved biosensor sensitivity with a 3 nM detection limit for hydrogen peroxide. While carbon paste has been found to be an attractive matrix for biosensor research because it can be doped with catalysts and biomolecules, Mailley significantly improved such amperometric biosensors by using a composite of carbon paste and in situgenerated polypyrrole containing the enzyme polyphenol oxidase for catechol detection (161). The composite exhibited much improved enzyme retention because of its effective entrapment by polypyrrole. Abad et al. introduced an immobilization technique to attach glycosylated proteins covalently to self-assembled monolayers on gold electrodes (162). Rather than using boronic acids, which form reversible bonds with saccharides, the authors combined such boronates with epoxy groups to achieve a very stable covalent linkage. A method to determine the concentration and isomer ratio of urocanic acid, which is important in understanding the photoimmunosupression in the skin, was developed by Tatsuma et al. by monitoring the inhibition of the hydrogen peroxide reduction at a heme peptide-modified electrode (163). Since the two different isomers show different inhibition of this response, the current before and after UV irradiation, which transforms the trans into the cis isomer, could be used to estimate the isomer ratio of the analyte. Mao et al. developed an enzyme-modified ring-disk carbon film electrode embedded in a thin-layer radial flow cell for the determination of trace amounts of hydrogen peroxide from brain microdialysate (164). While the ring electrode contained horseradish peroxidase for actual hydrogen peroxide detection, the disk electrode contained ascorbate oxidase to preoxidize and eliminate ascorbic acid that would otherwise interfere with the on-line analysis. In an alternate approach, Choi et al. explored the use of an insoluble oxidant membrane placed in front of an enzyme containing film to remove interfering oxidizable species (165). Creatinine and glucose biosensors were used as model systems, and the best oxidant was determined to be PbO2. Rather than using enzymes as biocatalysts to detect their substrates, Neufeld et al. electrochemically determined the enzymes released from lysed bacteria as a method to quantify and identify bacteria (166). In this example, a bacteriophage specific for Escherichia coli was used to release the bacterial cell content into solution. Amperometric detection of the marker enzyme activity (a galactosidase) gave detection limits as low as 1 colony-forming unit/100 mL sample. Sun and Jin determined zeptomole quantities of enzymes from individual human erythrocytes by electrokinetically injecting the sample into a capillary where the sample was electromigrated to a region of higher temperature to initiate enzyme reaction (167). The electroactive product NADH of the model enzyme glucose6-phosphate dehydrogenase used here was then monitored at a Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
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carbon fiber disk bundle electrode. This is one of many examples of a hyphenated technique where electrochemistry is used to inject, separate, and detect the analyte. Immunosensors. The principles of electrochemical immunosensors are now well established, and current developments go mainly in the direction of miniaturization and the fabrication of array systems in the form of biosensor chips and the exploration of alternate interrogation principles. The group of Fritsch reported on an immunoassay in a microcavity format containing a recessed microdisk with covalently attached antibody and a nanoband gold electrode for voltammetric detection of the enzyme label reaction product, p-aminophenol, with 56-zmol detection limits for the detection of IgG (168). In another example, Kojima et al. developed an electrochemical protein chip with an array of 36 platinum electrodes, in addition to thin-film silver/silver chloride electrodes and auxiliary electrodes, integrated on a glass substrate (169). Immobilization was achieved by plasma polymerization of a siloxane structure that showed no detectable nonspecific adsorption, and independent enzyme labeled sandwich immunoassays were successfully performed at different sites on the chip. The group of Smyth developed a competitive electrochemical enzyme-labeled immunoassay for sequential analyses of atrazine without any washing or regeneration steps (170). This was achieved by allowing the redox centers of the horseradish peroxidase enzyme label to couple directly to the conducting polymer substrate. Atrazine was detected down to 0.1 ppb concentrations. Grant et al. improved on an interesting label-free and reversible electrochemical immunosensor principle originally reported by Sadik and Wallace (171). The antibodies (against bovine serum albumin and digoxin) were embedded into conducting polypyrrole films and interrogated by pulsed amperometry. The chronoamperometric responses were reversible in quiescent solutions and showed a linear measuring range between 0 and 50 ppm. Dai et al. proposed the use of a pseudoreagentless amperometric immunosensor based on the direct electrochemistry of horseradish peroxidase (172). This enzyme was labeled to the antibody for the target antigen (carcinoma antigen-125), which were both deposited onto the sensor platform before measurement. When increasing concentrations of antigen were present in the sample, the current from the enzyme was found to decrease because of the competition between antigen present in the sample and immobilized on the electrode surface, yielding an apparently reagentless assay. Capacitance and impedance techniques are increasingly being used to probe immunoreactions at electrode surfaces, somewhat in analogy to surface plasmon resonance. Of course, as a labelfree technique, they are potentially very versatile but more prone to effects from nonspecific adsorption than established voltammetric techniques using an enzyme label, for example. The group of Sadik used differential impedance spectroscopy to monitor the kinetics and surface loading of protein immobilization and antibody-antigen reactions as a fundamental technique to understand surface deposition mechanisms and surface reactivity (173). Corry et al. also probed antibody-antigen binding events at gold-coated quartz crystals and indium-doped tin oxide films by electrochemical impedance spectroscopy (174). The electro3294
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chemical results were compared to surface roughness experiments using atomic force microscopy. Zayats et al. used impedance measurements on ion-sensitive field-effect transistor devices to determine the film thicknesses of the biomaterial, with good correlations to surface plasmon resonance measurements on the same system (175). This device is mainly useful for the detection of large analytes such as antibodies or the toxin cholera, for which an assay was developed. Oligonucleotides: Direct Detection. Numerous label-free detection methods have been explored in the past few years. De los Santos-Alvarez electrochemically oxidized the adenine bases of adsorbed oligonucleotides on pyrolytic graphite electrodes (176). They found that the reaction products were electroactive and strongly adsorbed onto the electrodes, which could be used to detect specific DNA sequences and synthetic homopolynucleotides. In a similar approach, Jelen et al. treated DNA with a strong acid to release its purine bases that were then detected by cathodic stripping voltammetry on a copper amalgam or hanging mercury drop electrode with subnanomolar detection limits (177). Ozkan et al. used the direct electrochemical oxidation of the guanine bases by differential pulse voltammetry at a carbon paste electrode to monitor the hybridization of DNA (178). Since peak currents for this assay were different for an allele-specific mutation, a labelfree yes/no system for the desired mutation was developed. The group of Mascini also used the electrochemical response of the guanine bases in the target DNA in a label-free assay but substituted all guanine bases in the immobilized probe DNA by inosine (179). This assay was developed in view of the detection of PCR samples of 244-base pair fragments related to the apolipoprotein E in just 10 min. Palecek’s group significantly reduced the problem of nonspecific adsorption of undesired nucleotides at the electrode surface by physically separating the recognition and detection surfaces (180). Hybridization was achieved at paramagnetic beads, followed by acid treatment that released adenine into solution that was detected at a mercury electrode. This sensitive label-free method was demonstrated with numerous types of oligonucleotides, and some possibilities to further increase the sensitivity through the use of catalytic schemes were discussed. See also Janata’s work above for yet another example of a label-free DNA detection method (67). Oligonucleotides: Intercalator Detection. The electrochemical detection of DNA via redox-active or electrocatalytic intercalators is an attractive approach to oligonucleotide hybridization measurements because the target DNA does not need to be chemically modified. For example, Maruyama et al. developed an osmium(II) complex containing amine electron-donating groups that showed a high binding affinity (3 × 107 M-1) to double stranded DNA and a low half-wave potential (181). When probe DNA was immobilized onto a gold electrode, detection limits for the electrochemical determination of target DNA was found to be 0.1 ng L-1 with a wide linear range. In a related approach, albeit not with a classical intercalator as reporter molecule, Masarik et al. proposed the adsorptive transfer stripping square wave voltammetric detection of streptavidin and avidin to quantify DNA hybridizations of biotinylated oligonucleotides (182). Detection limits were found to be as low as 6 pM for denatured streptavidin. Homberg and Thorp performed an electrochemical study and digital simulation to quantify the binding and rate
constants for the reaction of DNA with two different intercalators used simultaneously, one acting as an electrocatalyst for guanine oxidation, giving higher currents with higher double-stranded DNA concentrations, and the other used as redox probe, giving lower currents in the presence of DNA because of decreased mass transport (183). Wong and Gooding explored a mixed monolayer on gold containing single-stranded DNA and incubated with the redox-active intercalator 2,6-disulfonic acid anthraquinone for DNA detection (184). Only when complementary DNA was allowed to interact with the monolayer were voltammetric peaks for the oxidation and reduction of the intercalator observed, indicating that the double-stranded DNA was needed for electron transfer. Binding to DNA with mismatched base pairs gave reduced signals. Yang et al. used the polymerase chain reaction to amplify the desired DNA with 7-deaza analogues of guanine and adenine in order to obtain a larger electrochemical oxidation current in the presence of a ruthenium(II) bipyridine as electrocatalyst (185). Fahlman and Sen proposed molecular design strategies in order to use the change in electron-transfer properties of doublestranded DNA as an aptamer for the detection of intercalators, not the other way around (186). However, the selectivity of such a sensor was not yet characterized and is perhaps quite limited. Mugweru and Rusling developed a self-contained probe for damaged DNA with a catalytic film containing the DNA intercalator ruthenium-bipyridine and square wave voltammetric detection (187). When double-stranded DNA was subjected to the suspected carcinogen styrene oxide, the catalytic current was found to increase linearly with time. The mechanism of this assay was explained with the catalyst having improved access to the oxidizable bases of the damaged and partly unwound DNA, thereby increasing the current compared to undamaged DNA. In a related approach, Zhou et al. screened for DNA damage by forming a multilayer thin film containing the double-stranded DNA of interest and myoglobin or cytochrome P450 (188). Upon activation with acid, sample styrene was converted to the carcinogenic styrene oxide in situ by the enzyme and the intercalators ruthenium(II) and cobalt(II) bipyridine were used to electrochemically distinguish between intact and damaged DNA. Kelley’s group used the electrocatalytically enhanced voltammetric ruthenium(III) hexamine response to monitor DNA hybridization at a gold electrode (189). Since the ruthenium complex interacts electrostatically with DNA, it leads to a larger current when hybridized DNA is present. A single base pair mismatch could be identified by following the voltammetric response as a function of hybridization time. Yamashita et al. found in a detailed study that single base pair mismatches in DNA assays involving a 20mer probe can be electrochemically identified by using the intercalator ferrocenylnaphthalene diimide (190). Quartz crystal microbalance and MALDI-TOFLMS studies confirmed that the number of binding intercalator molecules decreased with increasing number of base pair mismatches. Oligonucleotides: Enzyme Amplified. Enzyme-amplified electrochemical oligonucleotide assays have been developed in analogy to earlier enzyme-labeled immunoassays. Aguilar and Fritsch introduced such an adaptation to a classical sandwich assay to detect Cryptosporidium parvum in water samples (191). The probe DNA was attached via its 5′-amine terminus to a selfassembled monolayer of mercaptoundecanoic acid on a gold
electrode. Upon hybridization with the target 121-nucleotide sequence, a secondary DNA probe tagged to alkaline phosphatase was hybridized. The enzyme generated aminophenyl phosphatase, from its added substrate p-aminophenol, which was detected electrochemically at the gold electrode. Detection limits were found to be ∼150 nM, with good selectivity. Kim et al. used the same enzyme reaction in a related DNA assay, but by using an aminated dendrimer containing ferrocenyl groups as electrocatalyst between the self-assembled monolayer and the DNA probe to increase sensitivity (192). The group of Heller used a carbon electrode chemically modified with a redox polymer and electrodeposited avidin to construct a sandwich assay, with probe DNA or RNA binding to the target, which in turn binds to an enzyme-labeled oligonucleotide delivered to the sample (193). The electrode was made specific by conjugating biotinylated probe RNA or DNA to the deposited avidin. Upon cohybridization with the target oligonucleotide and a horseradish peroxidase-tagged oligonucleotide, a sandwich was formed that was interrogated electrochemically by measuring the hydrogen peroxide reduction current. The electrical wiring of the enzyme with the redox polymer, which this group has already successfully used for glucose sensor development, is one of the key features of this electrochemical assay that takes ∼30 min to complete. Subsequent work of the same group used a microelectrode configuration for increased mass transport and achieved a 100-fold improvement in detection limit down to ∼20 pM levels (194). Williams et al. developed a related method for rapid DNA screening, using a very simple modified streptavidin carbon-polymer composite electrode that can be renewed by polishing between measurements (195). In this approach, target and probe DNA and a horseradish peroxidase enzyme label bound to a suitable antigen are all added to the sample at the same time, eliminating separate binding and washing steps. Willner’s group utilized an enzyme label for DNA detection that produces an insoluble reaction product (196). The readout was accomplished by impedance spectroscopy (and by a quartz crystal microbalance), and DNA detection limits were found to be on the order of 10-13 M. Oligonucleotides: Nanoparticles and Quantum Dots. Nanoparticle labels for oligonucleotides are known to be very attractive in spectroscopic readout methods and share unique properties that are very useful for electrochemical detection as well. In many cases, metal nanoparticles can be oxidized to form metal ions that are conveniently determined electrochemically. A recent example for this approach was described by Oxsoz et al., who monitored the direct oxidation current of gold nanoparticle tags upon hybridization of tagged target DNA and probe DNA covalently attached onto a graphite electrode (197). In another approach, the group of Wang used gold nanoparticles coated with ferrocenylhexanethiol and streptavidin (the latter for attachment of the biotinylated DNA probe) (198). Upon forming of a DNA sandwich, the ferrocene groups were detected electrochemically with a linear measuring range for DNA between 7 and 150 pM. The main advantage of this method lies in its experimental simplicity since no enzyme or enzyme substrate is needed and amplification is achieved by the large number of ferrocene groups present for each DNA binding event. Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
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Recently, such nanoparticle tags have been combined with magnetic particles for the purpose of additional preconcentration (199). Here, magnetic particles with probe DNA were used to capture target DNA, which in turn was allowed to hybridize with a secondary probe DNA tagged to a given metal nanoparticle. After hybridization, the ensembles were preconcentrated magnetically at an electrode and the nanoparticles were oxidized chemically and detected by anodic stripping voltammetry. The authors demonstrated simultaneous DNA assays with 0.3 nM detection limits by introducing up to three different nanoparticle tags (ZnS, CdS, and PbS) that could easily be electrochemically resolved. The same authors also introduced polystyrene beads containing defined amounts of various nanoparticles as electrochemical encoded tags in complete analogy to fluorescent polystyrene tags used in flow cytometry or random fiber-optic arrays (200). The electrochemical signatures were found to correlate well with the original nanoparticle loading concentrations. CONCLUSIONS The topic of electrochemical sensors is already quite vast and continues to grow and broaden. The field of potentiometric sensors, as a mature technology, has experienced important change in the past few years. The principal developments in this area focus on reducing the detection limit to true trace levels, down to the low parts per trillion concentration range, and there are important advances in the areas of materials and active components design. Importantly, potentiometry and the field of ion-transfer voltammetry start to approach each other to the extent that the design of instrumentally controlled ion-selective electrodes now becomes possible. Voltammetric sensor development focuses on further miniaturization, the reduction of the addressable sample volume, and the application to difficult in vivo and environmental sensing situations. Moreover, numerous materials characteristics are being improved to achieve improved selectivity as well as a larger potential window in aqueous samples. Electrochemical gas sensors are based on a wide range of mechanisms, ranging from simple resistance measurements to true electrochemical conversions at a three-phase interface. Developments in this area are quite divergent, with some researchers targeting the direct selective detection of analytes based on materials properties as well as the magnitude of the applied potential, others using an array of simpler, less selective systems in conjunction with a separation device such as a portable gas chromatograph, and yet others pursuing the concept of the electronic nose with an array of rather nonspecific sensors. It must be noted that the last concept has been rather successful for the distinction of individual gases or, at the most ternary mixtures, but normally fail at analyzing complex sample mixtures as they are often encountered in the real world. Electrochemical biosensor concepts are a vast area of research that continues to develop at a rapid pace. The enzyme-based biosensor is the classical biosensor, and the development of the glucose sensor is still the largest area of research, although it is very often used as a model system. Thermophilic enzymes for higher stability, improved materials for better biocompatibility, reduced interference, and improved enzyme stability, and the electronic wiring of enzymes to electrodes for mild and direct transduction of the signal are all important approaches that have 3296 Analytical Chemistry, Vol. 76, No. 12, June 15, 2004
been explored. Affinity sensors using DNA or immunological recognition units are perhaps not classical sensors because they lack in many cases reversibility. Yet, the number of electrochemical detection schemes for measuring these extremely important analytes is very inspiring. They range from direct label-free detection principles or intercalator-based techniques, the use of enzyme labels that form electroactive or insoluble products, to the application of quantum dots and magnetic particles as labels. In many of these detection principles, extremely low levels of detection with excellent selectivity and the capability of detecting single base pair mismatches has been demonstrated. Clearly, the area of electrochemical sensor research is very active and fruitful. It must be emphasized that many of the challenges that remain in some cases, especially in the area of selectivity, may be overcome by their integration into more complex analytical systems that combine online sampling and separation steps. However, in the cases where direct detection in unmodified samples is possible, the high analysis speed and the capability of detecting extremely small volumes without significantly perturbing the sample remain highly attractive characteristics of electrochemical sensors. ACKNOWLEDGMENT
This author gratefully acknowledges the National Institutes of Health and the Petroleum Research Fund (administered by the American Chemical Society) for supporting his research on electrochemical sensors. Eric Bakker is currently an Alumni Professor in the Department of Chemistry at Auburn University. After undergraduate and graduate studies of chemistry and analytical chemistry with the late Wilhelm Simon at the Swiss Federal Institute of Technology in Zurich, Switzerland, he pursued postdoctoral studies at the University of Michigan. He joined the faculty at Auburn University in 1995 as an Assistant Professor and was promoted to Associate Professor in 1998 and to full professor in 2003. His research interests include fundamental and applied aspects of potentiometric, voltammetric, and optical sensors based on molecular recognition and extraction principles. He has published about 120 papers in this field.
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