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further sample dilution and band broadening. Nevertheless this is currently the most popular method for LC-ECL and is the simpler to optimize. The addition of ECL reagents to the mobile phase is a simpler method in principle, provided the separation and ECL reaction can occur in the same mobile phase, the ECL reagents are not strongly bound by the column, and the efficiency of the separation and retention times are not adversely affected. The flow cells previously described are commonly used for post-LC-ECL detection, although cell volumes may need to be reduced.

Ru(bpy)32 ECL has been the most successfully coupled to chromatography as the reactions occur in a wide range of organic and aqueous solutions, and over a wide pH range. Indeed Ru(bpy)32 ECL is often enhanced by the presence of an organic modifier such as acetonitrile or methanol. Figure 8 shows an example of an HPLC separation with ECL detection of a mixture of glyphosate and related compounds by Ridlen et al. [61]. A novel indirect detection method for liquid chromatography based on the ECL of luminol has been reported by Wang and Yeung [62]. The mobile phase used contained luminol, hydrogen peroxide, and cobalt(II), which produces a steady ECL emission on passing through a postcolumn ECL flow cell. When an analyte is eluted, it displaces components of the ECL reaction thus decreasing the ECL emission and producing a negative peak. In principle a vast range of non-ECL-active analytes can be determined by this method, crucially those for which conventional ultraviolet absorbance is insensitive.

7. ECL IN CAPILLARY ELECTROPHORESIS

Over the past decade capillary electrophoresis (CE) has proved a rapid, efficient, and versatile method of separating analytes in extremely small sample volumes, with particular suitability for biochemical analysis. During this period several CE methods with chemiluminescence detection have been documented based on acridinium esters, luminol, peroxyoxalate, and firefly luciferase using a range of different apparatus. These have been reviewed by Garcı´a Campan˜a et al. [63]. More recently ECL detection has been coupled with CE. The flow cells previously described for FIA and LC analysis are, however, not ideally suitable for coupling to CE systems since their internal volumes would lead to excessive band broadening of the nanoliter volume analyte peaks. Several slightly different approaches to the development of a CE-ECL system have been reported for use with both Ru(bpy)32 [64–67] and luminol [68] ECL. An example of such a system is shown in Figure 9. In general, the working electrode is either a fine platinum wire or carbon fiber, which is placed very close to the end of the CE capillary. The end of the capillary and working electrode are placed within a reservoir holding a few milliliters of buffer forming the ECL cell, which is housed

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Figure 9 Schematic diagram for capillary electrophoresis with postcapillary ECL detection. (A) Overview of the apparatus. (B) Enlarged view of the etched joint and ECL detection cell. The PMT (not shown) is positioned directly over the Pt working electrode, and the entire apparatus placed inside a light-tight housing. (From Ref. 64.)

within a light-tight enclosure. Other conventional reference and counterelectrodes are also placed within the reservoir. There is obviously a need to isolate the high voltage used to drive the CE separation from the capillary output where the lowvoltage ECL detection occurs. To do this it is necessary to ground the capillary near the outlet end. This is achieved by making a section of the capillary porous, by etching or cracking, and encapsulating it in a buffer-filled reservoir that is connected to ground. The high-voltage circuit is then between the sample injection end of the capillary and this ground buffer reservoir. Bulk flow of the liquid then carries the separated analytes onto the ‘‘ECL cell.’’

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There are three main approaches to the introduction of the ECL reagents for CE. First is the addition of all necessary reagents to the eluent. This is the preferred method; however, as for HPLC, solution conditions have to be optimized to suit both the separation and the ECL reaction. Second, the ECL reagents may be mixed online with the CE stream at a mixing junction. However, this is not ideal, again owing to band broadening of the analyte peaks. Finally, the simplest solution is to add the necessary reagents to the ECL cell reservoir. The small volume of analytes yields a low ECL signal such that sensitive detectors (i.e., cooled PMTs) or photon-counting methods are often used. The geometry of the electrode arrangement is also far from ideal for efficient light collection, so parabolic mirrors are often employed for maximum light collection. Fiber optic bundles placed either side of the electrode have also been used, which carry the light to the phototransducer external to the cell. In addition, the buildup of analytes, reagents, and eluent in the ECL detection cell causes a significant background signal. The consequence of these factors is that only relatively high limits of detection are currently achievable of 10 5–10 7 M. However, since the sample volumes are generally so small, these can equate to mass detection limits of attomoles. Nevertheless with ECL moving increasingly toward biochemical applications, the design and construction of more robust and efficient CE-ECL detection cells should allow greater exploitation of CE in ECL analysis.

8. ENZYME-COUPLED ECL FOR BIOSENSING

Enzyme-coupled ECL enables the selective determination of many clinical analytes that are not in themselves directly electrochemiluminescent, but that can act as substrates for a variety of enzymic reactions. There are two general strategies for ECL: the use of dehydrogenase enzymes, which convert NAD to NADH, and oxidase enzymes, which produce hydrogen peroxide.

As can be seen in Figure 10, NADH contains a tertiary amine functional group, and has been determined by Ru(bpy)32 ECL to M levels [14, 69]. However, in NAD this group has been converted to an aromatic secondary amine, which gives virtually no ECL response. (Similarly it has been noted that NADPH and NADP show the same contrasting ECL activity.) This difference has been exploited for the determination of analytes such as glucose and ethanol, for which oxidation by the appropriate dehydrogenase enzyme is accompanied by the conversion of added NAD to NADH, which is subsequently detected by Ru(bpy)32 ECL. The ECL reaction of NADH and Ru(bpy)33 also regenerates NAD and Ru(bpy)32 . Similarly analytes that take part in enzymic reactions in which NADH is oxidized can also be detected, the ECL intensity being inversely proportional to the concentration of analyte. Bicarbonate has been determined in this way [70].

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Figure 10 Structures of NAD and NADH showing the part of the molecule where reversible reduction occurs, changing the ECL reactivity.

Hydrogen peroxide produced as a result of reactions of oxidase enzymes with analyte substrates can be sensitively determined, both directly by luminol ECL and indirectly by Ru(bpy)32 ECL. For the latter, hydrogen peroxide is detected on the basis of its ability to diminish the ECL reaction between Ru(bpy)32 and added oxalate, by reacting with, and depleting the concentration of, oxalate. Thus ECL intensity is inversely proportional to the concentration of analyte. This principle has been used, for example, to determine cholesterol [70].

In general, enzyme-coupled ECL produces detection limits in the 10 6– 10 8 M range, which are within or below the clinical range for typical analytes. Calibrations are often nonlinear since enzyme kinetics, ECL reactivity, and mass transfer effects, when immobilized reagents are used, all affect the sensitivity of the method. Ru(bpy)32 ECL is compatible with the optimum pH for many enzyme reactions, whereas luminol ECL occurs at a much higher pH. However, in biologically derived samples, many species interfere with Ru(bpy)32 ECL reactions, either constructively, such as oxalate, NADH, and other amines, or destructively, such as vitamin C, uric acid, and paracetamol. Hence sample pretreatment, the use of chromatographic separation, or dialysis through a membrane may be required.

In many FIA methods an inline immobilized enzyme reactor may be used. Alternatively the enzyme may be immobilized at the electrode surface. Wilson et al. [71] covalently attached glucose oxidase to an aminosilanized indium tin oxide (ITO)-coated glass wafer, which was used as the working electrode in a fountain flow cell. Light was recorded at the back of the transparent electrode. The cell was used for the luminol ECL determination of glucose to a limit of detection of 0.4 mM. Martin and Nieman [72] have used polymer entrapment to coimmobilize both an enzyme and Ru(bpy)32 to form a range of biosensors. Coimmobilization of both these components in the same film led to a much reduced enzyme activity; thus various multilayer configurations were tried. Ru(bpy)32 was absorbed into Nafion, while the enzyme was immobilized using a water-soluble Eastman AQ polymer. Stacking the two layers on top of each

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other gave the greatest sensitivity; however, the arrangement lacked stability and robustness. Preferred orientations were using adjacent layers such that the samples flowed first over the enzyme polymer and then over Ru(bpy)32 -polymer- coated electrode, or placing the Ru(bpy)32 -polymer-coated electrode and enzyme-polymer on opposite sides of a thin flow channel.

9. ECL IMMUNOASSAY AND DNA PROBES

The use of ECL detection for immunoassays has several advantages. No radioactive isotopes are used, thus reducing the problems of storage, handling, and disposal of samples and reagents. The labels are very stable with shelf lives in excess of 1 year at room temperature. Calibrations can be performed over a wide linear dynamic range, extending over up to six orders of magnitude in some cases. In addition, ECL labels based on Ru(bpy)32 can be very sensitively detected using an excess of an amine coreactant, and such labels are small allowing multiple labeling of an analyte without affecting its immunoreactivity [14–16].

The potential of ECL detection for immunoassays has led to the development of automated commercial instrumentation, first by IGEN and subsequently by Perkin-Elmer and Boehringer Mannheim GmbH. The latest commercial instruments use the following immunomagnetic method. Small magnetic beads are supplied coated with streptavidin to which biotin molecules are attached, in turn bound to a selected antibody, antigen, or DNA probe. These beads are combined with the analyte that binds to the immobilized group on the bead. Next Ru(bpy)32 -labeled antibodies or antigens, specific for the analyte, are added that bind to the analyte attached to the bead. In a flow-through system the beads are magnetically captured onto the working electrode, and the residual sample matrix and labels swept clear. ECL is then produced from the Ru(bpy)32 -based labels by applying a potential to the working electrode in the presence of an excess of tripropylamine. Rapid assays have been developed for a range of applications, including the diagnosis of thyroid diseases, tumors, infectious diseases, anemia, cardiological function, pregnancy, and sex function [73]. Figure 11 shows the basic principle of this technology.

Yu and Stopa have recently used an ECL immunoassay for the detection of virulent pathogenic bacteria in a range of environmental, food, and other biological samples, using Ru(bpy)32 -labeled antibodies [74]. Traditional methods for bacterial detection and identification are laborious and time consuming, typically taking 24–48 h to complete. The ECL method, however, was shown to detect Bacillus anthrax spores, Escherichia coli O157:H7, and Salmonella typhimurium to a limit of detection of 1000 cells/mL, in under 1 h, including sample preparation.

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Figure 11 The basic principle of ECL immunoassay using streptavidin-coated magnetic beads, and labels based on Ru(bpy)32 .

A few other ECL immunoassay and DNA probe methods have been designed. An enzymatic immunoassay based on luminol ECL has been developed by Wilson et al. for the detection of pesticides in drinking water to less than 0.1 ppb [75]. Transparent, aminosilanized, ITO-coated glass electrodes were derivatized with aminodextran covalently modified with atrazine caprioic acid. Antibodies to atrazine were labeled with glucose oxidase enzyme and used in an indirect competitive binding assay to detect atrazine in solution. Labeled antibodies that bound to the immobilized atrazine at the electrode surface were detected by an ECL reaction between oxidized luminol and H2 O2 produced by the enzyme with the addition of glucose. Kankare et al. demonstrated that oxide-bound terbium(III) can be excited at an aluminum electrode surface in aqueous solution to yield narrow-band terbium emissions (548 nm) in the presence of persulfate [76]. When chelated with 2,6-bis[N,N-bis(carboxymethyl)aminomethyl]-4-benzo- ylphenol, terbium cathodic luminescence had a significantly longer lifetime, such that time-resolved ECL was possible, the intensity measured a short time after electroexcitation, once short-lived background ECL had diminished. This terbium chelate has subsequently been used as an immunoassay label for the determination of human pancreatic phospholipase A2 to 10 ng/ml. Xu and Bard developed a biosensor based on single-stranded DNA immobilized on an electrode covered with alkanebisphosphonate, which could recognize a complimentary strand of DNA [77]. Hybridization of unlabeled DNA could be determined using ECL produced during the oxidation of Ru(1,10-phenanthroline)32 intercalated with

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DNA and tripropylamine. Alternatively, target DNA immobilized on the film could be determined by hybridization with a Ru(bpy)32 -labeled complementary DNA strand.

10. ECL IN MINIATURIZED ANALYTICAL SYSTEMS

Recently the miniaturization of analytical systems has received considerable interest, with the aim of realizing the whole analytical process of sampling, separation, and detection, incorporated on a single chip of perhaps a few square centimeters in area [78]. Such chips usually consist of multilayered structures of micromachined or etched glass, silicon, or polymer, with narrow interlocking channels for the passage and manipulation of reagents. These are moved either by external, conventional pumps or by electrokinetic means on the chip itself. ECL has potential as a detection system in such systems, since the components needed for ECL generation and detection are easily miniaturized. However, to date only a few devices using ECL have been reported [39, 79–81].

These miniature devices can be mounted close to a photodetector, but with a more elegant arrangement using photodiodes, the photodetector can become an intrinsic component of the device itself. This can be achieved either by bonding the photodiode to the exterior surface of a transparent window of the cell [80], or by direct fabrication in chips made from silicon substrates [81]. Plain platinum or silver metal pseudoreference electrodes that are readily miniaturized can replace more complex reference electrode constructions. Electrode formats used include fine wires, metal foils, or metal coatings typically 1 m thick. With use of transparent ITO electrodes [80] or interdigitated electrodes [81], ECL can be detected by an underlying photodetector while the top surface of the electrode is in contact with the test solution. In addition, since ECL and light emission is only stimulated in the direct vicinity of the electrode, they can be positioned such that only a small volume of the flow channel is probed at any one time. Hence it is possible to analyze extremely small volumes of solution. For example, Arora et al. used a multilayer polymer flow cell chip to detect Ru(bpy)32 with a limit of detection of 5 10 13 M in an effective cell volume of 100 nL, corresponding to just 30,000 molecules [39].

11.ECL IN ELECTRODE CHARACTERIZATION AND THE VISUALIZATION OF ELECTROCHEMICAL PROCESSES

Recently ECL has been utilized as a diagnostic tool to visualize electrochemical processes, to characterize electrode surfaces in terms of their uniformity or microarchitecture, and to reveal the coverage and heterogeneity of layers of com-

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pounds attached to the electrode surface. Much electrochemical data relies on the assumption that the observed current is representative of the electrode reaction at all parts of the electrode surface, which is uniformly active and accessible. ECL is being used to nondestructively probe electrodes where current density is not evenly distributed across the electrode. This effect may be due to absorbed species, oxide or other pacifying films, differential reactivities at various crystal faces at the electrode surface, analyte depletion downstream in a flow cell, diffusion effects, or the use of composite electrodes [82]. Equipment and procedures for the ECL visualization of faradic processes and electrode surface heterogeneity have been described by Kukoba and Rozhitskii [83].

The extent of coverage of electrodeposited films can be imaged by ECL to a resolution of about 0.5 m, a limit set by light microscopy and the lifetime of the excited species as they diffuse away from the electrode surface [83–85]. ECL is generally inhibited and absent from areas of the electrode covered by the film. Photographs give qualitative information of electrode activity, yet quantitative data can be obtained by tracking a detector, such as a PMT, across the electrode surface, or by using image capture on a CCD camera. Many ECL reactions have been used for imaging, depending on the electrode material or absorbate under study. Since luminol and Ru(bpy)32 are negatively and positively charged, respectively, they can be used to visualize different types of oxidation sites on an electrode. For example, Hopper and Kuhr demonstrated that surface oxide on carbon fiber microelectrodes inhibited luminol ECL while it facilitated Ru(bpy)32 ECL [85]. In organic solvents IAECL has been used, with rubrene or diphenylanthracene as the fluorescer [83].

ECL has also been produced from organized monolayers of Ru(bpy)32 , or its derivatives such as surfactants formed by using ligands with long-chain hydrocarbons, on a variety of electrode surfaces. Measurements can be used to determine absorption isotherms. Thiol derivatives have a particular affinity for gold electrodes and self-assemble into monolayers [14]. Although fairly intense ECL has been observed from absorbed monolayers of ECL active species, their current lack of robustness limits their analytical potential in terms of producing a sensor. However, by coupling with imaging techniques it should be possible to optically detect the arrival and interaction of small numbers of reactant species at the monolayer/solution interface [14].

12. CONCLUSIONS

In conclusion, ECL should continue to have a promising future in analytical science. The versatility of Ru(bpy)32 ECL will continue to be exploited for an increasingly diverse range of analytical determinations, especially in biomedical applications and immunoassay. Inevitably, for all but the most simple sample

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matrices, ECL will be coupled with a separation step such as HPLC or CE to remove interfering species; however, ECL is wholly compatible with these techniques. There are also good prospects for ECL to be used as a detection technique for miniaturized analytical systems. The production of a really robust and reproducible ECL sensor incorporating immobilized ECL reagents holds great potential but is yet to be fully realized, with further research required in this area. Finally, ECL should be a useful technique to nondestructively visualize electrochemical processes and electrode surfaces, and it is hoped that this will be further exploited in the future.

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