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Analytical applications of the electrochemiluminescence of tris (2,2′-bipyridyl) ruthenium and its derivatives
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Trends in Analytical Chemistry, Vol. 23, No. 6, 2004
Analytical applications of the electrochemiluminescence of tris (2,20-bipyridyl) ruthenium and its derivatives Xue-Bo Yin, Shaojun Dong, Erkang Wang This article presents the state of the art of analytical applications of the electrochemiluminescence (ECL) of tris (2,20 -bipyridyl) ruthenium (Ru(bpy)32þ ) and its derivatives. In the last seven years, Ru(bpy)32þ ECL has attracted much interest from analysts and been successfully exploited as a detector of flow injection analysis (FIA), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and micro total analysis systems (TAS). Immobilization of Ru(bpy)32þ on a solid surface provides several advantages over the solution-phase ECL procedure, such as the simplicity of experimental design and cost-effectiveness. After a brief discussion of the mechanism of Ru(bpy)32þ ECL, we discuss its applications in FIA, HPLC, CE and TAS and give special attention to the design of Ru(bpy)32þ ECL cells and some immobilization techniques of Ru(bpy)32þ ; we focus on papers published after 1997. ª 2004 Published by Elsevier B.V. Keywords: Capillary electrophoresis; Electrochemiluminescence; Flow injection analysis; High-performance liquid chromatography; Immobilization; Micro total analysis systems; Ru(bpy)32þ
1. Introduction Xue-Bo Yin, Shaojun Dong, Erkang Wang* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun 130022, China
*Corresponding author. Professor & Director of the Center, State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China. Tel.: +86 431-5262003; Fax: +86 431-5689711; E-mail: [email protected]
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Electrochemiluminescence (ECL), whereby electrochemically generated reactants undergo a high-energy electron transfer reaction to generate an excited state, has proved to be a powerful analytical tool that combines the simplicity of electrochemistry with the inherent sensitivity and the wide linear range of the chemiluminescence (CL) method. Tokel and Bard’s work [1] about the emission from the excited state of Ru(bpy)32þ formed via the electrochemical method started the application of the Ru(bpy)32þ ECL in analytical science. Since then, Ru(bpy)32þ ECL has become a sensitive detection method and Ru(bpy)32þ has also been one of the most studied and
exploited inorganic ECL compounds to date because of its stability and its capability of undergoing ECL at room temperature in aqueous buffered solutions. As described in previous reviews [2–5], Ru(bpy)32þ has been widely used in ECL flow cells and probe injection analysis systems to detect various chemicals and biochemical assays. Moreover, unlike some other CL reagents, which are consumed in the CL reaction, Ru(bpy)32þ is regenerated during the ECL process, as described the mechanism section below. As a result, a reagentless ECL sensor can be constructed by immobilizing the Ru(bpy)32þ on an electrode surface [2–5]. Compared with the solution-phase ECL procedure, the immobilization of the Ru(bpy)32þ makes experimental design simple and the cost low because there is no need to deliver extra Ru(bpy)32þ reagent [2–5]. Although Ru(bpy)32þ ECL detection has some benefits, there are some drawbacks, such as lack of selectivity and dependence on the environmental factors, e.g., viscosity [6], temperature, surfactant [7], ion strength, and pH. However, the lack of selectivity can be overcome by coupling Ru(bpy)32þ ECL with flow injection analysis (FIA), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and micro total analysis systems (lTAS), as discussed in the following sections. Ru(bpy)32þ ECL, as a detection method with detection limits of subpicomolar concentration and an extremely wide dynamic range, has been used successfully for the determination of
0165-9936/$ - see front matter ª 2004 Published by Elsevier B.V. doi:10.1016/S0165-9936(04)00603-X
Trends in Analytical Chemistry, Vol. 23, No. 6, 2004
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oxalate and some amine-containing analytes in FIA, HPLC, CE and lTAS. This article presents the state of the art of Ru(bpy)32þ ECL using the ECL mechanism and applying FIA, HPLC, CE, and lTAS for detection. We give special attention to the design of Ru(bpy)32þ ECL cells and focus on papers published after 1997.
2. Mechanism Generally, a Ru(bpy)32þ ECL reaction is initiated in three main ways. The first ECL reaction sequence is based on the annihilation of Ru(bpy)3þ and Ru(bpy)33þ , which are produced by applying a suitable voltage. When Ru(bpy)3þ reacts with Ru(bpy)33þ , the excited state Ru(bpy)32þ results, and that produces an orange emission centered on 610 nm. The above annihilation reaction can be carried out as redox-cycling procedure by using interdigitated carbon electrodes [8,9]. The original Ru(bpy)32þ species is regenerated in the following procedure: 2þ
e ! RuðbpyÞ3
2þ
þ e ! RuðbpyÞ3
RuðbpyÞ3 RuðbpyÞ3
3þ
þ
þ
3þ
2þ
RuðbpyÞ3 þ RuðbpyÞ3 2þ
RuðbpyÞ3
! RuðbpyÞ3 2þ
! RuðbpyÞ3
2þ
þ RuðbpyÞ3
þ hv
Ru(bpy)32þ
Another ECL reaction occurs by oxidizing Ru(bpy)32þ to produce Ru(bpy)32þ in the presence of a strong reducing agent. This mechanism is termed the oxidative-reduction mode. Probably the most efficient and most commonly used amine for this type of ECL reaction is tripropylamine (TPA). It has been proposed [10,11] that its oxidative-reduction ECL mechanism is as follows: e ! RuðbpyÞ3
3þ
þ NPr3 ! RuðbpyÞ3
RuðbpyÞ3
3þ
2þ
þ Pr3 Nþ
3þ
2þ
2þ
þ Pr2 Nþ ¼ CHEt
þ hv
Bard et al. [12] gave a new route, involving the TPrA cation radicals (TPrAþ ) as intermediates in the generation of Ru(bpy)32þ , which was established recently, based on the results of scanning electrochemical microscopy. This type of ECL was well documented and led to the recent development of highly sensitive detection schemes for most analytical applications. Since the mechanism of oxidative-reduction mode between Ru(bpy)32þ and
þ S2 O83
S2 O83 ! SO42 þ SO4 3þ
þ SO4 ! RuðbpyÞ3
þ
3þ
þ SO42 2þ
RuðbpyÞ3 þ RuðbpyÞ3 2þ
2þ
! RuðbpyÞ3
þ
RuðbpyÞ3
þ Pr2 NC HEt ! RuðbpyÞ3
2þ
RuðbpyÞ3
þ
þ e ! RuðbpyÞ3
RuðbpyÞ3 þ S2 O82 ! RuðbpyÞ3
RuðbpyÞ3
Pr3 Nþ ! Pr2 NC HEt þ H RuðbpyÞ3
2þ
RuðbpyÞ3
2þ
2þ
RuðbpyÞ3
amines involves an electron transfer from the amine to Ru(bpy)32þ , an inverse relationship exists between the first ionization potential of the non-bonding orbital of the amine and the ECL intensity. For alkylamines, the ECL intensity can be ordered as tertiary > secondary > primary, whereas the ionization potential of the alkylamines is ordered as primary > secondary > tertiary [2,9,11]. In the comprehensive study of Ru(bpy)32þ ECL process, the oxidative-reduction ECL mode was used to determine some analytes, as described in the following section. Some researchers were concerned to enhance the ECL intensity and thus the detection sensitivity. The effects of the working electrode and its surface hydrophobicity on the emission intensity of Ru(bpy)32þ /TPA system were studied recently [9,13,14]. The hydrophobicity of the Pt and Au surface resulting from the adsorption of surfactant significantly enhanced both TPA oxidation current and ECL intensity [13,14]. However, the surfactant suppression of both TPA oxidation and ECL emission at a glass carbon electrode indicated that the surfactant effect depended on the electrode material [13,14]. The enhancement effect of halide ions on the emission intensity of the Ru(bpy)32þ /TPA system was presumed to be because the halide ions inhibited the growth of surface oxides on Pt and Au electrodes and led to an increase TPA oxidation current [10]. The excited species Ru(bpy)32þ is formed by the reduction of Ru(bpy)32þ in the presence of a strong oxidizing reagent within the third, so-called reductiveoxidation mode. The ECL reaction of peroxidisulphate (S2 O82 ) with Ru(bpy)32þ is as follows [15]:
! RuðbpyÞ3 2þ
! RuðbpyÞ3
2þ
þ RuðbpyÞ3
þ hv
The above three modes, involving the direct electrontransfer between the reagent and working electrode, are carried out either very positive potential (1.2 V versus Ag/AgCl) or very negative potential ()2.0 V versus Ag/AgCl) of the working electrode [2–5]. We found an entirely different approach to generating cathodic Ru(bpy)32þ ECL triggered by reactive oxygen species (ROS) [16]. Two reduction waves were observed in cyclic voltammograms (CVs) at a 4-mm radius glassy carbon working electrode in an air-saturated phosphate buffer (pH 6.8) containing 1.0 mM Ru(bpy)32þ , while the first wave starting at )0.4 V was attributed to the http://www.elsevier.com/locate/trac
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reduction of dissolved oxygen. The latter wave between )1.13 and )1.18 V, just before the onset of water reduction, was attributed to the reduction of Ru(bpy)32þ . Interestingly, light emission appeared at the onset of oxygen reduction ()0.4 V) and the highest point was reached at )0.8 V, and it then decreased slightly beyond )0.8 V. In the presence of TPA, while the reduction current of oxygen did not seem to change, light emission increased dramatically. Considering the strong oxidizability of ROS, we propose that cathodic ECL is initiated by ROS. ROS oxidizes Ru(bpy)32þ to Ru(bpy)33þ . In the presence of TPA, Ru(bpy)33þ oxidizes TPA to form a reducing radical, which reacts with another Ru(bpy)33þ to produce Ru(bpy)32þ . The latter produces the orange emission. For a comparison of anodic ECL (initiated at +1.2 V) and the novel cathodic ECL (initiated at )0.8 V) [16], the intensity of the cathodic ECL for 10 lM TPA being 0.94 times of the anodic ECL indicates that the sensitivity of cathodic ECL for TPA was similar to that of anodic ECL.
3. Application of Ru(bpy)32 + ECL in FIA For Ru(bpy)32þ ECL in FIA, analyte and Ru(bpy)32þ , if not immobilized on solid support, flow continuously and are mixed together either in a mixing tee placed before the detection cell or in a reaction/detection cell. Since the concentration of analyte in the carrier stream is not constant with time, the signal is obtained when the analyte is transported by the carrier into the reaction/ detection region. Li et al. [17,18] proposed a FI–ECL cell comprising a PTFE cell body and a cover window, as shown in Fig. 1. Platinum foil imbedded in the center facing the glass window was used as working electrode. Between the Pt foil and the glass window, a piece of O-ring was used as the spacer, ensuring a thin-layer thickness of ca. 0.1 mm and the thin-layer volume of 17.7 ll. The stainless steel outlet tube and an Ag/AgCl wire served as counter and reference electrodes, respectively. Adrenaline [17], noradrenaline and dopamine [18] were detected with the FI–ECL system using their inhibition by Ru(bpy)32þ / TPA ECL systems, as shown in Fig. 2. Another flow cell used for FI–ECL measurement was assembled from a piece of epoxy-plate-supported Pt electrode, a polyethylene spacer and a piece of glass window with 162 ll cell volume. Two strong clamps were used to hold the components of ECL cell tight together [19]. Because a microcell can be mounted much closer to the photomultiplier tube (PMT), development of a miniaturized ECL flow cell has aroused most researchers’ interest. Recently, a miniaturized chip-type Ru(bpy)32þ detection cell was developed by our laboratory [20]. As shown in Fig. 3, the cell was fabricated from two pieces of glass using wet chemical etching. A stainless steel 434
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Figure 1. Diagram of the FI–ECL flow-through thin-layer cell: (a) above the cell body; (b) below the cover window. Adapted from [17].
Figure 2. Schematic diagram of the FI–ECL system. Adapted from [18].
tube (acting as both working electrode and capillary guide) and silver wire were glued in working electrode and reference electrode accommodating groove. Another silver wire, inserted into a channel, acted as quasireference electrode. The bottom plate also acted as the observation window. With this design, the dead volume was increasingly decreased to only the volume of the etched channel. It was successfully used to detect oxalate, TPA, and proline with FI mode. The additional advantage of this chip-type ECL cell design over conventional FI–ECL cell is that it can be used as CE detector after suitably modifying it because of its low internal volume [20]. 4. Application of Ru(bpy)32 + ECL in HPLC Some factors may affect the successful coupling of HPLC with ECL detection. First, solvent compatibility between
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Figure 3. Schematic view of chip-type flow cell: (a) Design pattern of flow channel and accommodating groove; (b) the overall schematic view of chip-type flow cell. (1) Cover plate; (2) bottom plate; (3) pattern design of flow channel; (4) working electrode; (5) reference electrode groove; (6) capillary groove; (7) flow channel; (8) working electrode groove; (9) capillary groove; (10) reference electrode groove; (11) Ru(bpy)32þ solution reservoir; (12) waste reservoir; (13) working electrode guide; (14) capillary guide; (15) accommodated at the bottom of (10); (16) connecting capillary; (17) working electrode; (19) silver-wire quasi-reference electrode; (20) inlet capillary; (21) outlet tubing; and, (22) rubber septa seal. Adapted and modified from [20].
the HPLC separation and ECL detection must be considered. The effect of the organic solvent on ECL intensity depends on the ECL system employed. A variety of organic solvents have been used for Ru(bpy)32þ ECL in HPLC. The use of acetonitrile and methanol was proved to have no detrimental effect on ECL intensity. Second, the quantum efficiency of Ru(bpy)32þ ECL should be examined, since the ECL intensity depends on ECL systems and reaction conditions, such as pH, flow rate and the configuration of the flow cell. If the pH of separation buffer and detection solution differ, a post-column pH adjustment is required. Because of the good compatibility of Ru(bpy)32þ ECL with common HPLC modes, most of papers on HPLC–ECL are focused on the designs of flow cells and analytical application [21–28] (see Table 1).
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Different approaches were utilized to deliver Ru(bpy)32þ to the systems. Ru(bpy)32þ can be oxidized to Ru(bpy)33þ by bulk electrolysis and then post-column mixed with the HPLC effluent [22]. After derivatization with emetine dithiocarbamate, Tsukagoshi et al. [22] used Ru(bpy)33þ HPLC–ECL to detect Cu(II) and Co(II); they used the Ru(bpy)33þ resulting from Ru(bpy)32þ oxidation in an electrochemical reactor with electrolytic current of 150 lA at the carbon electrode. But Ru(bpy)33þ is unstable; an alternative is a post-column addition of Ru(bpy)32þ into the HPLC effluent stream and on-line oxidation from Ru(bpy)32þ to Ru(bpy)33þ . The reaction is triggered directly in the reaction cell [20–27]. A post-column Ru(bpy)32þ addition to reversed-phase chromatographic effluent and a commercially available spiral CE flow cell were utilized to detect fatty acids and primary amines after derivatization [23,24]. This in situ generation of Ru(bpy)33þ has the advantage of better reproducibility compared with the direct addition of Ru(bpy)33þ . With similar instrumentation, Park et al. [25] applied a post-column Ru(bpy)32þ addition in the HPLC–ECL system to the determination of b-blocker in human plasma and urine. The above two methods [22–26] may result in the dilution of the analytes and band broadening. An alternative method is to add Ru(bpy)32þ pre-column to the mobile phase of HPLC if Ru(bpy)32þ does not significantly interfere with the separation [26]. But the procedure by which the HPLC column was flushed with the mobile phase containing Ru(bpy)32þ for 1 h to saturate the column with Ru(bpy)32þ was wasteful of time and reagent [26]. Investigation of the difference between Ru(bpy)32þ pre-column and post-column addition was carried out; Fig. 4 shows the typical schematic diagrams of both modes [26]. Immobilization of Ru(bpy)32þ on the surface of electrode can overcome the above problem [28]. Choi et al. [28] reported that Ru(bpy)32þ was immobilized on an electrode surface comprising sol–gel-derived titania and Nafion for the determination of erythromycin in human urine sample. To obtain high sensitivity in HPLC–ECL systems, it is necessary to consider constructing an ECL flow cell. As
Table 1. Application of Ru(bpy)32þ ECL in HPLC Analyte
Separation mode
Matrix
Addition of Ru(bpy)32 +
Limit of detection
Electrochemical system
References
Lupin alkaloids Cu(II), Co(II)
Ion pair Reversed phase
Standard Standard
Post-column Post-column
3–11 1011 g/ml 0.65, 17 pg/10 ll
[21] [22]
Fatty acid histamine Dansyl-Glu
Reversed phase Reversed phase, ion pair Reversed phase
Human plasma Plasma
45–70, 70 fmol 0.1, 0.2 lM
[23] [26]
Juice
Post-column Pre-column, post-column Post-column
Three-electrode Ru(bpy)33þ was added Not given Three-electrode
107 M
Three-electrode
[27]
Reversed phase
Human urine
Immobilization
1 lM
Three-electrode
[28]
Ascorbic dehydro-ascorbic acid Erythromycin
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sealed in a Struers Epofix HQ two-component epoxy resin polymerized in a suitable mould. The saturated calomel electrode (SCE) reference electrode was externally connected to the cell via the outlet tube. A 200-lm spacer created the void volume of the thin-layer flow. 0.5 mM Ru(bpy)32þ , as post-column solution, was mixed with chromatographic effluent by a tee junction.
5. Application of Ru(bpy)32 + ECL in CE
Figure 4. Schematic diagram for: (a) Ru(bpy)32þ mobile phase addition detection; (b) Ru(bpy)32þ post-column addition detection: A, silica pre-column; B, guard column. Adapted from [26].
in FI–ECL systems, most works concerning HPLC–ECL have used thin-layer flow cell. Zorzi et al. [27] built an ECL thin-layer flow cell, which comprises a threeelectrode amperometric cell. The working and counter electrodes, together with inlet and outlet pipes, were
Unlike HPLC coupling with ECL detection, the electric current in CE capillaries under high electric field affects the ECL procedure on the microelectrode, and the low flow rate of CE makes it more difficult to couple ECL directly to CE. Since ECL involves the formation of electronically excited states of electrochemically generated species on the microelectrode, a special design of ECL detector for CE is often necessary, so that the effect of CE high electric field on ECL procedure must be eliminated or decreased to some extent (see Table 2). An electric-field decoupler has been used to isolate ECL detection from the CE high voltage field. Forbes et al. [29] used a porous polymer junction near the end of the capillary to complete the CE circuit and inserted a working electrode into the capillary end to generate ECL. A parabolic reflector was used to direct emitted light
Table 2. Application of Ru(bpy)32þ ECL in CE and lTAS Analyte
Separation channel (lm)
Matrix
Field decoupler
Limit of detection
Electrochemical system
References
Proline Val Phe Diphen-hydramine Tramadol Lidocaine Procyclidine Proline histidine TPA Proline TPA Lidocaine TPA
75
Standard solution
Yes
Two-electrode system
[30]
25 25
Rabbit plasma urine Urine
No No
Three-electrode system Three-electrode system
[31] [32]
25 50 21 75
Urine Standard solution Standard solution Urine
No No No No
Three-electrode system DC battery DC battery (1.5 V) Three-electrode system
[33] [34] [35] [36]
75
Standard solution
No
0.2 lM 0.7 lM 0.5 lM 2 108 M 6 108 M 4.5 108 M 1 109 M 1 lM 2–5 lM 5 1011 M (TPA) 2 108 M (Lidocaine) 1 lM (TPA)
Three-electrode system with immobilizing Ru(bpy)32þ on working electrode
[37]
75
No
1 mM (Proline) Not given
Three-electrode system
[38]
50
Degradation solution of pHPPa Standard solution
No
1.2, 50, 25 lM
Three-electrode system
[39]
60 20 chip
Standard solution
No
5 106 M
Voltage required to obtain ECL reaction from the electric field in separation channel
[43]
15 48
Standard solution
No
6 106 M 1.2 lM
Three-electrode system
[44]
Proline Oxalic acid Pro, Valine, Phenylalanine Ru(bpy)32þ
Ru(phen)32þ Proline a
pHPP: p-hydroxyphenylpyruvic acid.
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to the PMT. Wang and Bobbitt [30] separated the electrophoretic current from the electrochemical current via an on-column fracture, which was covered with a Nafion tube. But the analytes may diffuse through the fracture, thus inducing zone broadening and sample leakage. If a capillary with a small inner diameter (625 lm) or a low conductivity buffer was used, no significant effect of high electric field on ECL detection was observed. With a small inner diameter capillary CE–ECL system, our group developed some methods for the determination of diphenhydramine [31], tramadol and lidocaine [32], and procyclidine [33]. To obtain high ECL efficiency, the relative position between the working electrode and the end of capillary must be carefully adjusted to perform in situ oxidation of Ru(bpy)32þ to Ru(bpy)33þ . While some have aligned the microelectrode with the capillary tip [31–33], others have directly inserted the microelectrode into the capillary end [29,30]. Chiang et al. [34,35] used an indium/tin oxide (ITO)coated glass plate, positioned at the capillary end, as the working electrode for in situ generation of active Ru(bpy)33þ . The CE effluent directly impinges on the surface of the ITO electrode and the potential of the ITO electrode was controlled using a DC battery, which eliminates the need for an on-column field decoupler. The emitted ECL was collected with an optic fiber attached to the back of the ITO electrode plate. The first kind of cell body was made of polytetrafluoroethylene (PTFE), which also functioned as a cathodic buffer reservoir for CE, and the ITO electrode was held in the cell body by another piece of Teflon plate and four screws [34]. To simplify configuration of the ECL cell and to improve CE efficiency, they reported a simple, cost-effective CE–ECL system [35]. The detector was constructed by gluing a 0.5 ml plastic sample vial onto ITO-coating glass plate and at right angles to it. The CE efficiency is significantly improved (from 4000 to >105 theoretical plates/m), perhaps because less turbulence was generated at the capillary outlet region in the new configuration [35]. We also investigated the influence of CE current on the ECL process without an electric field decoupler by using a 75-lm i.d. capillary and a high-conductivity CE buffer [36]. The hydrodynamic CV and corresponding Ru(bpy)32þ ECL under the high-voltage field showed that the electrophoretic current did not annihilate the Ru(bpy)32þ ECL, but made the ECL potential shift more positively. Using what we had learned above, we developed a convenient end-column ECL detector without the electric field decoupler [36]. As shown in Fig. 5 [36], the end of the separation capillary was inserted into a stainless steel tube that served as the CE ground electrode. To align the CE capillary with the working electrode, the latter was adjusted and fixed by three screws from three directions. To avoid blocking photon detection by the PMT, the ref-
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Figure 5. Schematic diagram of the ECL cell coupled with separation capillary for end-column mode. (1) Stainless-steel holding screw; (2) PVC capillary holder; (3) stainless-steel tube; (4) separation capillary; (5) reference electrode; (6) counter electrode; (7) nylon screws for alignment; (8) working electrode cable; (9) PVC electrode holder; (10) working electrode; (11) optical glass window; (12) PMT. Adapted from [36].
erence electrode and the counter electrode were inserted into the solution above the working electrode. The volume of cell was about 300 ll. However, the reservoir design was limited in its application, as the concentration of Ru(bpy)32þ in the reservoir was found to change with time because of dilution and evaporation, which affected both sensitivity and reproducibility of ECL response. We also developed a CE-solid ECL detection system by using large inner diameter capillary without decoupler [37], to compare it with solution ECL detection. Although the solid ECL system gives high detection limits, the solid sensor is cost-effective and regenerative. Fabrication of a solid ECL detector only consumed 0.1 ll of 10 mM Ru(bpy)32þ , which was less than the hundreds of ll of Ru(bpy)32þ in the general solution CE–ECL system [37]. Based on the above model, a commercial CE–ECL system was recently fabricated in our laboratory; it integrated CE high-voltage power together with the electrochemical and ECL system, and included data acquisition and data treatment. As shown in Fig. 6, all operations were computer-controllable, and CE current, electrochemical current, electrochemical potential and ECL intensity versus time are presented simultaneously. With this system, the study of electrochemistry, ECL and CE–ECL processes can be progressed. Recently, Chen et al. [38] also proposed a CE–ECL system, where a Pt wire (100 lm diameter, 10 cm long) was wound around the outer surface of the separation capillary. Epoxy resin coating on the wound Pt wire was used to insulate most of the Pt wire except for the surface http://www.elsevier.com/locate/trac
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Figure 6. A commercial CE–ECL system fabricated by our laboratory.
near the outlet of the capillary. A Pt ring electrode was obtained by polishing the outlet surface and that gave a flat surface opposite to the detection window. The Pt ring electrode CE–ECL system has two apparent advantages: • the surface of the working electrode can be placed opposite to the detection window, resulting in nearly 100% of ECL emission being detectable by PMT, • it has a very small dead volume because the working electrode can be located as closely as possible to the detection window.
sample injection, and simple instrumentation set-up facilitating integration [40–42]. ECL based on Ru(bpy)32þ completely satisfies the above requirements. Arora et al. [43] described a chip CE–ECL device structure, as shown in Fig. 7. The glass devices measuring 5 cm 2 cm were fabricated using a standard photolithography procedure followed by wet chemical etching and thermal bonding. The total construction included a separation channel, an injection channel and a double-T injector. A ‘‘U’’-shaped Pt electrode was micro-fabricated within a glass device across the separation channel, where its legs functioned as floating
In contrast with ECL detection in HPLC and FIA, the addition of the precursor Ru(bpy)32þ to the capillary end using a pump and a mixing tee is little used [30,39] and Ru(bpy)32þ is often added to a post-capillary reservoir, incorporated in the capillary effluent [31–36,38]. Huang et al. [39] designed a CE end-column ECL detection system, where a syringe pump was used to deliver Ru(bpy)32þ solution. When combined with a falling-drop sample-introduction interface and a short capillary (6.8 cm), they obtained a sample throughput of 50/h [39]. A novel reagentless solid ECL detector has been fabricated by immobilizing Ru(bpy)32þ in a polymer film coated onto a Pt disk electrode in our laboratory [37]. 6. Application of Ru(bpy)32 + ECL in lTAS Miniaturization of conventional analytical equipment offers many advantages, such as short analysis time, low consumption of reactants, ease of automation, and integration of separation and detection on the same chip [40,41]. To utilize the full potential of miniaturized analytical instruments, there are strict requirements on the detectors, such as high sensitivity through low 438
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Figure 7. (a) Schematic diagram of the bonded glass chip, (b) channel layout, (c) photo of the device after drilling the holes and gluing the reservoirs. 1, Sample reservoir; 2, buffer reservoir; 3, sample waste; 4, buffer waste; 5, double-T injector; 6, separation channel; 7, floating platinum electrode; 8, sample filled into double-T injector; 9, vacuum; 10, direction of plug during separation. Adapted from [43].
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(a)
ITO electrode
unused reservoir buffer reservoir electrode plate
separation channel PMDS layer
sample reservoir
(b)
detection reservoir
ITO electrode
separation channel
The effective part (0.2 mm wide) containing with solution
Figure 8. Schematic of a microchip CE–ECL device: (a) top view of the PDMS layer and electrode plate; (b) an enlargement of the detection region. Adapted from [44].
working electrode and counter electrode. The voltage required to carry out the ECL reaction on the floating electrode was the result of the electric field presented in the separation channel during separation. Recently, our laboratory proposed an integrated indium tin oxide electrode-based Ru(bpy)32þ ECL detector for microchip CE device [44]. As illustrated in Fig. 8, this system utilized an ITO-coated glass slide as the chip substrate with a photolithographically fabricated ITO electrode located at the end of the separation channel. The separation and injection channels were contained in poly(dimethylsiloxane) (PDMS) layer, which was reversibly bound to the ITO electrode plate. With this construction, the alignment of the separation channel with the working electrode was greatly simplified and the photon-capturing efficiency enhanced [44].
7. Immobilization of the Ru(bpy)32 + on a solid surface The limitation of consuming expensive Ru(bpy)32þ can be overcome by immobilizing Ru(bpy)32þ in solid formats. Immobilization of Ru(bpy)32þ has several advantages over solution-phase ECL, such as simple experimental design and cost-effectiveness because there is no need to deliver extra Ru(bpy)32þ reagent. Directly synthesizing the polymer-containing Ru reagent at the surface is one mode of immobilization. Fresen et al. [45] developed a solid ECL sensor based on a poly[RuL3 ]2þ (where L ¼ vbpy or ester-substituted bipyridine) film on an array electrode or a sandwich-type arrangement. Light emission resulted from the reaction
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of RuL3þ and RuL33þ , which formed via independent control of the potentials of the closely spaced electrodes. Forster and Hogan [46] developed a new kind of electropolymerized [Ru(PVP-bpy)3 ]2þ -modified solid ECL electrode. But their analytical application was limited by the inhibition of mass transport within the polymer formed [45,46]. As a cation, Ru(bpy)32þ can be immobilized on some ion-exchange materials. Lin et al. [47] directly immobilized the Ru(bpy)32þ on a cationic ion-exchange resin and used it to determine oxalate, ethanol, and sulfite. The incorporation of Ru(bpy)32þ within the cationexchange polymer Nafion to fabricate an ECL-based sensor is still needed to improve reactivity and long-term stability [48]. Khramov and Collinson [49] reported the ECL of Ru(bpy)32þ ion-exchanged in Nafion–silica composite films, where the relatively slow diffusion of electroactive cations into Nafion can be improved by using Nafion–silica nanocomposites. We also reported the immobilization of Ru(bpy)32þ in the less hydrophobic PSS–silica (where PSS ¼ poly (sodium 4-styrene sulfonate) composite films [37,50,51], which were used as a solid ECL detector of CE [37] or electrostatically attached to the benzene sulfonic acid monolayer film [52]. Those ECL sensors showed good storage stability for six months. Eastman-AQ polymers are similar to Nafion in that they also contain sulfonated cation-exchange sites, but they are more hydrophilic than Nafion. Moreover, with their low cost, strong adherence to the surface, rapid response as well as permselective, ion-exchanging and anti-fouling properties, Eastman-AQ polymers appear to be attractive materials for sensor development. We incorporated AQ55D into silica sols and the resultant composite materials were used to immobilize Ru(bpy)32þ [53]. Compared with PSS, AQ55D does not need surfactant to prevent cracking of the composite film [53]. Choi et al. [28] reported an alternative matrix of composite films comprising sol–gel-derived titania and Nafion for the immobilization of Ru(bpy)32þ on an electrode surface and used it in HPLC detection. Modifying suitable material with Ru(bpy)32þ reagent has been used for the immobilization of Ru(bpy)32þ . Zhao et al. [54] reported on modified Ru(bpy)32þ chitosan coating on the Pt electrode. With this method, the response to trimethylamine was 39 times as weak as that obtained with a bare Pt electrode in an aqueous solution where Ru(bpy)32þ coexisted because of the electrostatic repulsion of the chitosan membrane having a positive charge to trimethylamine [54]. Compared with those methods, SiO2 -based gel is an attractive material because of its good three-dimension physical structure and high chemical stability, which allow the analyte to diffuse freely in the sol–gel film; but Ru(bpy)32þ leaching from the sol–gel film is the main drawback of this method [55]. It http://www.elsevier.com/locate/trac
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was also found that different co-reactants had different response times as a result of their different molecular structures, thus the diffusion of co-reactants differed [55]. Layer-by-layer (LBL) is an effective, attractive method often used for thin-film construction of polymers with oppositely charged colloidal particles. Dennany et al. [56] demonstrated direct ECL involving DNA in 10-nm films of cationic polymer [Ru(bpy)2 (PVP)10 ]2þ -assembled LBL with DNA. We have also used the simple, universal LBL technique to immobilize Ru(bpy)32þ [57]. Pre-made SiO2 nanoparticles, charged negatively at suitable pH, and Ru(bpy)32þ charged positively were used to form an ultrathin multilayer film via the LBL processing scheme, which was based on electrostatic interaction between positively charged Ru(bpy)32þ and negatively charged silica nanoparticles. After the [SiO2 /Ru(bpy)32þ ]n multilayer film was modified on ITO electrodes, we achieved high electroactivity, high stability and high reproducibility because of the high surface activity of the nanoparticles present [57]. 8. Conclusions ECL based on Ru(bpy)32þ has been successfully exploited as a sensitive detection technique for the determination of oxalate and a variety of amine-containing compounds in flowing streams, such as FIA, HPLC, CE and lTAS. Since Ru(bpy)32þ ECL emission is generated in situ in the reaction/detection cell with the original Ru(bpy)32þ species regenerated during the reaction, it does not need an external excitation light source and additional instrumentation to cut out scattered light for the excitation source, so sample dilution and band broadening are eliminated in a simple instrumental set-up. More applications of Ru(bpy)32þ ECL are required so that its inherent characteristics can be further exploited. We used Ru(bpy)32þ ECL to study the perchloratetriggered ion-gate behavior of a supported lipid bilayer membrane, which showed ion-gate behavior for the permeation of Ru(bpy)32þ in the presence of the perchlorate anion [58]. Zhan et al. [59,60] have devised a two-channel microfluidic sensor using anodic ECL as a photonic detector of cathodic redox reactions. The dual-electrode sensor relied on electrochemical detection at one electrode and ECL detection at the other. This approach allows the ECL reaction to be physically and chemically decoupled from the sensing channel of the device, which greatly expands the numbers of analytes that can be detected without derivatization. Acknowledgements This work is supported by the National Natural Science Foundation of China with Grant Nos. 20299030 and 440
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20335040 and National Key Basic Research Programs 2001CB5102 and 2002CB513110. X.B.Y. acknowledges the support of the China Postdoctoral Science Foundation for this project. References [1] N.E. Tokel, A.J. Bard, J. Am. Chem. Soc. 94 (1972) 2862. [2] R.D. Gerardi, N.W. Barnett, S.W. Lewis, Anal. Chim. Acta 378 (1999) 1. [3] W.Y. Lee, Mikrochim. Acta 127 (1997) 19. [4] K.A. F€ ahnrich, M. Pravda, G.G. Guilbault, Talanta 54 (2001) 531. [5] A.V. Kukoba, A.I. Bykh, I.B. Svir, Fresenius’ J. Anal. Chem. 368 (2000) 439. [6] A.M. Scott, R. Pyati, J. Phys. Chem. B 105 (2001) 9011. [7] S. Workman, M.M. Richter, Anal. Chem. 72 (2000) 5556. [8] G.C. Fiaccabrino, M. Koudelka-Hep, Y.T. Hsueh, S.D. Collins, R.L. Smith, Anal. Chem. 70 (1998) 4157. [9] P. Szrebowaty, A. Kapturkiewicz, Chem. Phys. Lett. 328 (2000) 160. [10] Y. Zu, A.J. Bard, Anal. Chem. 72 (2000) 3223. [11] F. Kanoufi, Y. Zu, A.J. Bard, J. Phys. Chem. B 105 (2001) 210. [12] W.J. Miao, J.P. Choi, A.J. Bard, J. Am. Chem. Soc. 124 (2002) 14478. [13] Y. Zu, A.J. Bard, Anal. Chem. 73 (2001) 3960. [14] F. Li, Y. Zu, Anal. Chem. 76 (2004) 1768. [15] T. Ala-Kleme, S. Kulmala, L. Vare, P. Juhala, M. Helin, Anal. Chem. 71 (1999) 5538. [16] W.-D. Cao, G.-B. Xu, Z.-L. Zhang, S.-J. Dong, Chem. Commun. 14 (2002) 1540. [17] F. Li, H. Cui, X.-Q. Lin, Anal. Chim. Acta 471 (2002) 187. [18] F. Li, Y.-Q. Pang, X.-Q. Lin, H. Cui, Talanta 59 (2003) 627. [19] C.-Y. Wang, H.-J. Huang, Anal. Chim. Acta 498 (2003) 61. [20] J.-F. Liu, J.-L. Yan, X.-R. Yang, E.-K. Wang, Anal. Chem. 75 (2003) 3637. [21] X. Chen, C.-Q. Yi, M.-J. Li, X. Lu, Z. Li, P.-W. Li, X.-R. Wang, Anal. Chim. Acta 466 (2002) 79. [22] K. Tsukagoshi, K. Miyamoto, R. Nakajima, N. Ouchiyam, J. Chromatogr. A 919 (2001) 331. [23] H. Morita, M. Konishi, Anal. Chem. 74 (2002) 1584. [24] H. Morita, M. Konishi, Anal. Chem. 75 (2003) 940. [25] Y.J. Park, D.W. Lee, W.Y. Lee, Anal. Chim. Acta 471 (2002) 51. [26] D.R. Skotty, W.Y. Lee, T.A. Nieman, Anal. Chem. 68 (1996) 1530. [27] M. Zorzi, P. Pastore, F. Magno, Anal. Chem. 72 (2000) 4934. [28] H.N. Choi, S.H. Cho, W.Y. Lee, Anal. Chem. 75 (2003) 4250. [29] G.A. Forbes, T.A. Nieman, J.V. Sweedler, Anal. Chim. Acta 347 (1997) 289. [30] X. Wang, D.R. Bobbitt, Anal. Chim. Acta 383 (1999) 213. [31] J.-F. Liu, W.-D. Cao, X.-R. Yang, E.-K. Wang, Talanta 59 (2003) 453. [32] W.-D. Cao, J.-F. Liu, H.-B. Qiu, X.-R. Yang, E.-K. Wang, Electroanalysis (NY) 14 (2002) 1571. [33] X.-H. Sun, J.-F. Liu, W.-D. Cao, X.-R. Yang, E.-K. Wang, Y.-S. Fung, Anal. Chim. Acta 470 (2002) 137. [34] M.-T. Chiang, C.-W. Whang, J. Chromatogr. A 934 (2001) 59. [35] M.-T. Chiang, M.-C. Lu, C.-W. Whang, Electrophoresis 24 (2003) 3033. [36] W.-D. Cao, J.-F. Liu, X.-R. Yang, E.-K. Wang, Electrophoresis 23 (2002) 3683.
Trends in Analytical Chemistry, Vol. 23, No. 6, 2004 [37] W.-D. Cao, J.-B. Jia, X.-R. Yang, S.-J. Dong, E.-K. Wang, Electrophoresis 23 (2002) 3692. [38] G.-N. Chen, Y.-W. Chi, X.-P. Wu, J.-P. Duan, N.-B. Li, Anal. Chem. 75 (2003) 6602. [39] X.-J. Huang, S.-L. Wang, Z.-L. Fang, Anal. Chim. Acta 456 (2002) 167. [40] D.R. Reyes, D. Iossifidis, P.-A. Auroux, A. Manz, Anal. Chem. 74 (2002) 2623. [41] P.-A. Auroux, D. Iossifidis, D.R. Reyes, A. Manz, Anal. Chem. 74 (2002) 2637. [42] J.-L. Yan, Y. Du, J.-F. Liu, W.-D. Cao, X.-H. Sun, W.-H. Zhou, X.-R. Yang, E.-K. Wang, Anal. Chem. 75 (2003) 5406. [43] A. Arora, J.C.T. Eijkel, W.E. Morf, A. Manz, Anal. Chem. 73 (2001) 3282. [44] H.-B. Qiu, J.-L. Yan, X.-H. Sun, J.-F. Liu, W.-D. Cao, X.-R. Yang, E.-K. Wang, Anal. Chem. 75 (2003) 5435. [45] D.A. Fresen, T. Kajita, D. Danielson, T.J. Meyer, Inorg. Chem. 38 (1999) 3442. [46] R.J. Forster, C.F. Hogan, Anal. Chem. 72 (2000) 5576. [47] J.-M. Lin, F. Qu, M. Yamada, Anal. Bioanal. Chem. 374 (2002) 1159. [48] Z. Hu, C.J. Seliskar, W.R. Heineman, Anal. Chem. 70 (1998) 5230.
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Analytical applications of the electrochemiluminescence of tris (2,20-bipyridyl) ruthenium and its derivatives Xue-Bo Yin, Shaojun Dong, Erkang Wang This article presents the state of the art of analytical applications of the electrochemiluminescence (ECL) of tris (2,20 -bipyridyl) ruthenium (Ru(bpy)32þ ) and its derivatives. In the last seven years, Ru(bpy)32þ ECL has attracted much interest from analysts and been successfully exploited as a detector of flow injection analysis (FIA), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and micro total analysis systems (TAS). Immobilization of Ru(bpy)32þ on a solid surface provides several advantages over the solution-phase ECL procedure, such as the simplicity of experimental design and cost-effectiveness. After a brief discussion of the mechanism of Ru(bpy)32þ ECL, we discuss its applications in FIA, HPLC, CE and TAS and give special attention to the design of Ru(bpy)32þ ECL cells and some immobilization techniques of Ru(bpy)32þ ; we focus on papers published after 1997. ª 2004 Published by Elsevier B.V. Keywords: Capillary electrophoresis; Electrochemiluminescence; Flow injection analysis; High-performance liquid chromatography; Immobilization; Micro total analysis systems; Ru(bpy)32þ
1. Introduction Xue-Bo Yin, Shaojun Dong, Erkang Wang* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun 130022, China
*Corresponding author. Professor & Director of the Center, State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China. Tel.: +86 431-5262003; Fax: +86 431-5689711; E-mail: [email protected]
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Electrochemiluminescence (ECL), whereby electrochemically generated reactants undergo a high-energy electron transfer reaction to generate an excited state, has proved to be a powerful analytical tool that combines the simplicity of electrochemistry with the inherent sensitivity and the wide linear range of the chemiluminescence (CL) method. Tokel and Bard’s work [1] about the emission from the excited state of Ru(bpy)32þ formed via the electrochemical method started the application of the Ru(bpy)32þ ECL in analytical science. Since then, Ru(bpy)32þ ECL has become a sensitive detection method and Ru(bpy)32þ has also been one of the most studied and
exploited inorganic ECL compounds to date because of its stability and its capability of undergoing ECL at room temperature in aqueous buffered solutions. As described in previous reviews [2–5], Ru(bpy)32þ has been widely used in ECL flow cells and probe injection analysis systems to detect various chemicals and biochemical assays. Moreover, unlike some other CL reagents, which are consumed in the CL reaction, Ru(bpy)32þ is regenerated during the ECL process, as described the mechanism section below. As a result, a reagentless ECL sensor can be constructed by immobilizing the Ru(bpy)32þ on an electrode surface [2–5]. Compared with the solution-phase ECL procedure, the immobilization of the Ru(bpy)32þ makes experimental design simple and the cost low because there is no need to deliver extra Ru(bpy)32þ reagent [2–5]. Although Ru(bpy)32þ ECL detection has some benefits, there are some drawbacks, such as lack of selectivity and dependence on the environmental factors, e.g., viscosity [6], temperature, surfactant [7], ion strength, and pH. However, the lack of selectivity can be overcome by coupling Ru(bpy)32þ ECL with flow injection analysis (FIA), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and micro total analysis systems (lTAS), as discussed in the following sections. Ru(bpy)32þ ECL, as a detection method with detection limits of subpicomolar concentration and an extremely wide dynamic range, has been used successfully for the determination of
0165-9936/$ - see front matter ª 2004 Published by Elsevier B.V. doi:10.1016/S0165-9936(04)00603-X
Trends in Analytical Chemistry, Vol. 23, No. 6, 2004
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oxalate and some amine-containing analytes in FIA, HPLC, CE and lTAS. This article presents the state of the art of Ru(bpy)32þ ECL using the ECL mechanism and applying FIA, HPLC, CE, and lTAS for detection. We give special attention to the design of Ru(bpy)32þ ECL cells and focus on papers published after 1997.
2. Mechanism Generally, a Ru(bpy)32þ ECL reaction is initiated in three main ways. The first ECL reaction sequence is based on the annihilation of Ru(bpy)3þ and Ru(bpy)33þ , which are produced by applying a suitable voltage. When Ru(bpy)3þ reacts with Ru(bpy)33þ , the excited state Ru(bpy)32þ results, and that produces an orange emission centered on 610 nm. The above annihilation reaction can be carried out as redox-cycling procedure by using interdigitated carbon electrodes [8,9]. The original Ru(bpy)32þ species is regenerated in the following procedure: 2þ
e ! RuðbpyÞ3
2þ
þ e ! RuðbpyÞ3
RuðbpyÞ3 RuðbpyÞ3
3þ
þ
þ
3þ
2þ
RuðbpyÞ3 þ RuðbpyÞ3 2þ
RuðbpyÞ3
! RuðbpyÞ3 2þ
! RuðbpyÞ3
2þ
þ RuðbpyÞ3
þ hv
Ru(bpy)32þ
Another ECL reaction occurs by oxidizing Ru(bpy)32þ to produce Ru(bpy)32þ in the presence of a strong reducing agent. This mechanism is termed the oxidative-reduction mode. Probably the most efficient and most commonly used amine for this type of ECL reaction is tripropylamine (TPA). It has been proposed [10,11] that its oxidative-reduction ECL mechanism is as follows: e ! RuðbpyÞ3
3þ
þ NPr3 ! RuðbpyÞ3
RuðbpyÞ3
3þ
2þ
þ Pr3 Nþ
3þ
2þ
2þ
þ Pr2 Nþ ¼ CHEt
þ hv
Bard et al. [12] gave a new route, involving the TPrA cation radicals (TPrAþ ) as intermediates in the generation of Ru(bpy)32þ , which was established recently, based on the results of scanning electrochemical microscopy. This type of ECL was well documented and led to the recent development of highly sensitive detection schemes for most analytical applications. Since the mechanism of oxidative-reduction mode between Ru(bpy)32þ and
þ S2 O83
S2 O83 ! SO42 þ SO4 3þ
þ SO4 ! RuðbpyÞ3
þ
3þ
þ SO42 2þ
RuðbpyÞ3 þ RuðbpyÞ3 2þ
2þ
! RuðbpyÞ3
þ
RuðbpyÞ3
þ Pr2 NC HEt ! RuðbpyÞ3
2þ
RuðbpyÞ3
þ
þ e ! RuðbpyÞ3
RuðbpyÞ3 þ S2 O82 ! RuðbpyÞ3
RuðbpyÞ3
Pr3 Nþ ! Pr2 NC HEt þ H RuðbpyÞ3
2þ
RuðbpyÞ3
2þ
2þ
RuðbpyÞ3
amines involves an electron transfer from the amine to Ru(bpy)32þ , an inverse relationship exists between the first ionization potential of the non-bonding orbital of the amine and the ECL intensity. For alkylamines, the ECL intensity can be ordered as tertiary > secondary > primary, whereas the ionization potential of the alkylamines is ordered as primary > secondary > tertiary [2,9,11]. In the comprehensive study of Ru(bpy)32þ ECL process, the oxidative-reduction ECL mode was used to determine some analytes, as described in the following section. Some researchers were concerned to enhance the ECL intensity and thus the detection sensitivity. The effects of the working electrode and its surface hydrophobicity on the emission intensity of Ru(bpy)32þ /TPA system were studied recently [9,13,14]. The hydrophobicity of the Pt and Au surface resulting from the adsorption of surfactant significantly enhanced both TPA oxidation current and ECL intensity [13,14]. However, the surfactant suppression of both TPA oxidation and ECL emission at a glass carbon electrode indicated that the surfactant effect depended on the electrode material [13,14]. The enhancement effect of halide ions on the emission intensity of the Ru(bpy)32þ /TPA system was presumed to be because the halide ions inhibited the growth of surface oxides on Pt and Au electrodes and led to an increase TPA oxidation current [10]. The excited species Ru(bpy)32þ is formed by the reduction of Ru(bpy)32þ in the presence of a strong oxidizing reagent within the third, so-called reductiveoxidation mode. The ECL reaction of peroxidisulphate (S2 O82 ) with Ru(bpy)32þ is as follows [15]:
! RuðbpyÞ3 2þ
! RuðbpyÞ3
2þ
þ RuðbpyÞ3
þ hv
The above three modes, involving the direct electrontransfer between the reagent and working electrode, are carried out either very positive potential (1.2 V versus Ag/AgCl) or very negative potential ()2.0 V versus Ag/AgCl) of the working electrode [2–5]. We found an entirely different approach to generating cathodic Ru(bpy)32þ ECL triggered by reactive oxygen species (ROS) [16]. Two reduction waves were observed in cyclic voltammograms (CVs) at a 4-mm radius glassy carbon working electrode in an air-saturated phosphate buffer (pH 6.8) containing 1.0 mM Ru(bpy)32þ , while the first wave starting at )0.4 V was attributed to the http://www.elsevier.com/locate/trac
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reduction of dissolved oxygen. The latter wave between )1.13 and )1.18 V, just before the onset of water reduction, was attributed to the reduction of Ru(bpy)32þ . Interestingly, light emission appeared at the onset of oxygen reduction ()0.4 V) and the highest point was reached at )0.8 V, and it then decreased slightly beyond )0.8 V. In the presence of TPA, while the reduction current of oxygen did not seem to change, light emission increased dramatically. Considering the strong oxidizability of ROS, we propose that cathodic ECL is initiated by ROS. ROS oxidizes Ru(bpy)32þ to Ru(bpy)33þ . In the presence of TPA, Ru(bpy)33þ oxidizes TPA to form a reducing radical, which reacts with another Ru(bpy)33þ to produce Ru(bpy)32þ . The latter produces the orange emission. For a comparison of anodic ECL (initiated at +1.2 V) and the novel cathodic ECL (initiated at )0.8 V) [16], the intensity of the cathodic ECL for 10 lM TPA being 0.94 times of the anodic ECL indicates that the sensitivity of cathodic ECL for TPA was similar to that of anodic ECL.
3. Application of Ru(bpy)32 + ECL in FIA For Ru(bpy)32þ ECL in FIA, analyte and Ru(bpy)32þ , if not immobilized on solid support, flow continuously and are mixed together either in a mixing tee placed before the detection cell or in a reaction/detection cell. Since the concentration of analyte in the carrier stream is not constant with time, the signal is obtained when the analyte is transported by the carrier into the reaction/ detection region. Li et al. [17,18] proposed a FI–ECL cell comprising a PTFE cell body and a cover window, as shown in Fig. 1. Platinum foil imbedded in the center facing the glass window was used as working electrode. Between the Pt foil and the glass window, a piece of O-ring was used as the spacer, ensuring a thin-layer thickness of ca. 0.1 mm and the thin-layer volume of 17.7 ll. The stainless steel outlet tube and an Ag/AgCl wire served as counter and reference electrodes, respectively. Adrenaline [17], noradrenaline and dopamine [18] were detected with the FI–ECL system using their inhibition by Ru(bpy)32þ / TPA ECL systems, as shown in Fig. 2. Another flow cell used for FI–ECL measurement was assembled from a piece of epoxy-plate-supported Pt electrode, a polyethylene spacer and a piece of glass window with 162 ll cell volume. Two strong clamps were used to hold the components of ECL cell tight together [19]. Because a microcell can be mounted much closer to the photomultiplier tube (PMT), development of a miniaturized ECL flow cell has aroused most researchers’ interest. Recently, a miniaturized chip-type Ru(bpy)32þ detection cell was developed by our laboratory [20]. As shown in Fig. 3, the cell was fabricated from two pieces of glass using wet chemical etching. A stainless steel 434
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Figure 1. Diagram of the FI–ECL flow-through thin-layer cell: (a) above the cell body; (b) below the cover window. Adapted from [17].
Figure 2. Schematic diagram of the FI–ECL system. Adapted from [18].
tube (acting as both working electrode and capillary guide) and silver wire were glued in working electrode and reference electrode accommodating groove. Another silver wire, inserted into a channel, acted as quasireference electrode. The bottom plate also acted as the observation window. With this design, the dead volume was increasingly decreased to only the volume of the etched channel. It was successfully used to detect oxalate, TPA, and proline with FI mode. The additional advantage of this chip-type ECL cell design over conventional FI–ECL cell is that it can be used as CE detector after suitably modifying it because of its low internal volume [20]. 4. Application of Ru(bpy)32 + ECL in HPLC Some factors may affect the successful coupling of HPLC with ECL detection. First, solvent compatibility between
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Figure 3. Schematic view of chip-type flow cell: (a) Design pattern of flow channel and accommodating groove; (b) the overall schematic view of chip-type flow cell. (1) Cover plate; (2) bottom plate; (3) pattern design of flow channel; (4) working electrode; (5) reference electrode groove; (6) capillary groove; (7) flow channel; (8) working electrode groove; (9) capillary groove; (10) reference electrode groove; (11) Ru(bpy)32þ solution reservoir; (12) waste reservoir; (13) working electrode guide; (14) capillary guide; (15) accommodated at the bottom of (10); (16) connecting capillary; (17) working electrode; (19) silver-wire quasi-reference electrode; (20) inlet capillary; (21) outlet tubing; and, (22) rubber septa seal. Adapted and modified from [20].
the HPLC separation and ECL detection must be considered. The effect of the organic solvent on ECL intensity depends on the ECL system employed. A variety of organic solvents have been used for Ru(bpy)32þ ECL in HPLC. The use of acetonitrile and methanol was proved to have no detrimental effect on ECL intensity. Second, the quantum efficiency of Ru(bpy)32þ ECL should be examined, since the ECL intensity depends on ECL systems and reaction conditions, such as pH, flow rate and the configuration of the flow cell. If the pH of separation buffer and detection solution differ, a post-column pH adjustment is required. Because of the good compatibility of Ru(bpy)32þ ECL with common HPLC modes, most of papers on HPLC–ECL are focused on the designs of flow cells and analytical application [21–28] (see Table 1).
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Different approaches were utilized to deliver Ru(bpy)32þ to the systems. Ru(bpy)32þ can be oxidized to Ru(bpy)33þ by bulk electrolysis and then post-column mixed with the HPLC effluent [22]. After derivatization with emetine dithiocarbamate, Tsukagoshi et al. [22] used Ru(bpy)33þ HPLC–ECL to detect Cu(II) and Co(II); they used the Ru(bpy)33þ resulting from Ru(bpy)32þ oxidation in an electrochemical reactor with electrolytic current of 150 lA at the carbon electrode. But Ru(bpy)33þ is unstable; an alternative is a post-column addition of Ru(bpy)32þ into the HPLC effluent stream and on-line oxidation from Ru(bpy)32þ to Ru(bpy)33þ . The reaction is triggered directly in the reaction cell [20–27]. A post-column Ru(bpy)32þ addition to reversed-phase chromatographic effluent and a commercially available spiral CE flow cell were utilized to detect fatty acids and primary amines after derivatization [23,24]. This in situ generation of Ru(bpy)33þ has the advantage of better reproducibility compared with the direct addition of Ru(bpy)33þ . With similar instrumentation, Park et al. [25] applied a post-column Ru(bpy)32þ addition in the HPLC–ECL system to the determination of b-blocker in human plasma and urine. The above two methods [22–26] may result in the dilution of the analytes and band broadening. An alternative method is to add Ru(bpy)32þ pre-column to the mobile phase of HPLC if Ru(bpy)32þ does not significantly interfere with the separation [26]. But the procedure by which the HPLC column was flushed with the mobile phase containing Ru(bpy)32þ for 1 h to saturate the column with Ru(bpy)32þ was wasteful of time and reagent [26]. Investigation of the difference between Ru(bpy)32þ pre-column and post-column addition was carried out; Fig. 4 shows the typical schematic diagrams of both modes [26]. Immobilization of Ru(bpy)32þ on the surface of electrode can overcome the above problem [28]. Choi et al. [28] reported that Ru(bpy)32þ was immobilized on an electrode surface comprising sol–gel-derived titania and Nafion for the determination of erythromycin in human urine sample. To obtain high sensitivity in HPLC–ECL systems, it is necessary to consider constructing an ECL flow cell. As
Table 1. Application of Ru(bpy)32þ ECL in HPLC Analyte
Separation mode
Matrix
Addition of Ru(bpy)32 +
Limit of detection
Electrochemical system
References
Lupin alkaloids Cu(II), Co(II)
Ion pair Reversed phase
Standard Standard
Post-column Post-column
3–11 1011 g/ml 0.65, 17 pg/10 ll
[21] [22]
Fatty acid histamine Dansyl-Glu
Reversed phase Reversed phase, ion pair Reversed phase
Human plasma Plasma
45–70, 70 fmol 0.1, 0.2 lM
[23] [26]
Juice
Post-column Pre-column, post-column Post-column
Three-electrode Ru(bpy)33þ was added Not given Three-electrode
107 M
Three-electrode
[27]
Reversed phase
Human urine
Immobilization
1 lM
Three-electrode
[28]
Ascorbic dehydro-ascorbic acid Erythromycin
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sealed in a Struers Epofix HQ two-component epoxy resin polymerized in a suitable mould. The saturated calomel electrode (SCE) reference electrode was externally connected to the cell via the outlet tube. A 200-lm spacer created the void volume of the thin-layer flow. 0.5 mM Ru(bpy)32þ , as post-column solution, was mixed with chromatographic effluent by a tee junction.
5. Application of Ru(bpy)32 + ECL in CE
Figure 4. Schematic diagram for: (a) Ru(bpy)32þ mobile phase addition detection; (b) Ru(bpy)32þ post-column addition detection: A, silica pre-column; B, guard column. Adapted from [26].
in FI–ECL systems, most works concerning HPLC–ECL have used thin-layer flow cell. Zorzi et al. [27] built an ECL thin-layer flow cell, which comprises a threeelectrode amperometric cell. The working and counter electrodes, together with inlet and outlet pipes, were
Unlike HPLC coupling with ECL detection, the electric current in CE capillaries under high electric field affects the ECL procedure on the microelectrode, and the low flow rate of CE makes it more difficult to couple ECL directly to CE. Since ECL involves the formation of electronically excited states of electrochemically generated species on the microelectrode, a special design of ECL detector for CE is often necessary, so that the effect of CE high electric field on ECL procedure must be eliminated or decreased to some extent (see Table 2). An electric-field decoupler has been used to isolate ECL detection from the CE high voltage field. Forbes et al. [29] used a porous polymer junction near the end of the capillary to complete the CE circuit and inserted a working electrode into the capillary end to generate ECL. A parabolic reflector was used to direct emitted light
Table 2. Application of Ru(bpy)32þ ECL in CE and lTAS Analyte
Separation channel (lm)
Matrix
Field decoupler
Limit of detection
Electrochemical system
References
Proline Val Phe Diphen-hydramine Tramadol Lidocaine Procyclidine Proline histidine TPA Proline TPA Lidocaine TPA
75
Standard solution
Yes
Two-electrode system
[30]
25 25
Rabbit plasma urine Urine
No No
Three-electrode system Three-electrode system
[31] [32]
25 50 21 75
Urine Standard solution Standard solution Urine
No No No No
Three-electrode system DC battery DC battery (1.5 V) Three-electrode system
[33] [34] [35] [36]
75
Standard solution
No
0.2 lM 0.7 lM 0.5 lM 2 108 M 6 108 M 4.5 108 M 1 109 M 1 lM 2–5 lM 5 1011 M (TPA) 2 108 M (Lidocaine) 1 lM (TPA)
Three-electrode system with immobilizing Ru(bpy)32þ on working electrode
[37]
75
No
1 mM (Proline) Not given
Three-electrode system
[38]
50
Degradation solution of pHPPa Standard solution
No
1.2, 50, 25 lM
Three-electrode system
[39]
60 20 chip
Standard solution
No
5 106 M
Voltage required to obtain ECL reaction from the electric field in separation channel
[43]
15 48
Standard solution
No
6 106 M 1.2 lM
Three-electrode system
[44]
Proline Oxalic acid Pro, Valine, Phenylalanine Ru(bpy)32þ
Ru(phen)32þ Proline a
pHPP: p-hydroxyphenylpyruvic acid.
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to the PMT. Wang and Bobbitt [30] separated the electrophoretic current from the electrochemical current via an on-column fracture, which was covered with a Nafion tube. But the analytes may diffuse through the fracture, thus inducing zone broadening and sample leakage. If a capillary with a small inner diameter (625 lm) or a low conductivity buffer was used, no significant effect of high electric field on ECL detection was observed. With a small inner diameter capillary CE–ECL system, our group developed some methods for the determination of diphenhydramine [31], tramadol and lidocaine [32], and procyclidine [33]. To obtain high ECL efficiency, the relative position between the working electrode and the end of capillary must be carefully adjusted to perform in situ oxidation of Ru(bpy)32þ to Ru(bpy)33þ . While some have aligned the microelectrode with the capillary tip [31–33], others have directly inserted the microelectrode into the capillary end [29,30]. Chiang et al. [34,35] used an indium/tin oxide (ITO)coated glass plate, positioned at the capillary end, as the working electrode for in situ generation of active Ru(bpy)33þ . The CE effluent directly impinges on the surface of the ITO electrode and the potential of the ITO electrode was controlled using a DC battery, which eliminates the need for an on-column field decoupler. The emitted ECL was collected with an optic fiber attached to the back of the ITO electrode plate. The first kind of cell body was made of polytetrafluoroethylene (PTFE), which also functioned as a cathodic buffer reservoir for CE, and the ITO electrode was held in the cell body by another piece of Teflon plate and four screws [34]. To simplify configuration of the ECL cell and to improve CE efficiency, they reported a simple, cost-effective CE–ECL system [35]. The detector was constructed by gluing a 0.5 ml plastic sample vial onto ITO-coating glass plate and at right angles to it. The CE efficiency is significantly improved (from 4000 to >105 theoretical plates/m), perhaps because less turbulence was generated at the capillary outlet region in the new configuration [35]. We also investigated the influence of CE current on the ECL process without an electric field decoupler by using a 75-lm i.d. capillary and a high-conductivity CE buffer [36]. The hydrodynamic CV and corresponding Ru(bpy)32þ ECL under the high-voltage field showed that the electrophoretic current did not annihilate the Ru(bpy)32þ ECL, but made the ECL potential shift more positively. Using what we had learned above, we developed a convenient end-column ECL detector without the electric field decoupler [36]. As shown in Fig. 5 [36], the end of the separation capillary was inserted into a stainless steel tube that served as the CE ground electrode. To align the CE capillary with the working electrode, the latter was adjusted and fixed by three screws from three directions. To avoid blocking photon detection by the PMT, the ref-
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Figure 5. Schematic diagram of the ECL cell coupled with separation capillary for end-column mode. (1) Stainless-steel holding screw; (2) PVC capillary holder; (3) stainless-steel tube; (4) separation capillary; (5) reference electrode; (6) counter electrode; (7) nylon screws for alignment; (8) working electrode cable; (9) PVC electrode holder; (10) working electrode; (11) optical glass window; (12) PMT. Adapted from [36].
erence electrode and the counter electrode were inserted into the solution above the working electrode. The volume of cell was about 300 ll. However, the reservoir design was limited in its application, as the concentration of Ru(bpy)32þ in the reservoir was found to change with time because of dilution and evaporation, which affected both sensitivity and reproducibility of ECL response. We also developed a CE-solid ECL detection system by using large inner diameter capillary without decoupler [37], to compare it with solution ECL detection. Although the solid ECL system gives high detection limits, the solid sensor is cost-effective and regenerative. Fabrication of a solid ECL detector only consumed 0.1 ll of 10 mM Ru(bpy)32þ , which was less than the hundreds of ll of Ru(bpy)32þ in the general solution CE–ECL system [37]. Based on the above model, a commercial CE–ECL system was recently fabricated in our laboratory; it integrated CE high-voltage power together with the electrochemical and ECL system, and included data acquisition and data treatment. As shown in Fig. 6, all operations were computer-controllable, and CE current, electrochemical current, electrochemical potential and ECL intensity versus time are presented simultaneously. With this system, the study of electrochemistry, ECL and CE–ECL processes can be progressed. Recently, Chen et al. [38] also proposed a CE–ECL system, where a Pt wire (100 lm diameter, 10 cm long) was wound around the outer surface of the separation capillary. Epoxy resin coating on the wound Pt wire was used to insulate most of the Pt wire except for the surface http://www.elsevier.com/locate/trac
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Figure 6. A commercial CE–ECL system fabricated by our laboratory.
near the outlet of the capillary. A Pt ring electrode was obtained by polishing the outlet surface and that gave a flat surface opposite to the detection window. The Pt ring electrode CE–ECL system has two apparent advantages: • the surface of the working electrode can be placed opposite to the detection window, resulting in nearly 100% of ECL emission being detectable by PMT, • it has a very small dead volume because the working electrode can be located as closely as possible to the detection window.
sample injection, and simple instrumentation set-up facilitating integration [40–42]. ECL based on Ru(bpy)32þ completely satisfies the above requirements. Arora et al. [43] described a chip CE–ECL device structure, as shown in Fig. 7. The glass devices measuring 5 cm 2 cm were fabricated using a standard photolithography procedure followed by wet chemical etching and thermal bonding. The total construction included a separation channel, an injection channel and a double-T injector. A ‘‘U’’-shaped Pt electrode was micro-fabricated within a glass device across the separation channel, where its legs functioned as floating
In contrast with ECL detection in HPLC and FIA, the addition of the precursor Ru(bpy)32þ to the capillary end using a pump and a mixing tee is little used [30,39] and Ru(bpy)32þ is often added to a post-capillary reservoir, incorporated in the capillary effluent [31–36,38]. Huang et al. [39] designed a CE end-column ECL detection system, where a syringe pump was used to deliver Ru(bpy)32þ solution. When combined with a falling-drop sample-introduction interface and a short capillary (6.8 cm), they obtained a sample throughput of 50/h [39]. A novel reagentless solid ECL detector has been fabricated by immobilizing Ru(bpy)32þ in a polymer film coated onto a Pt disk electrode in our laboratory [37]. 6. Application of Ru(bpy)32 + ECL in lTAS Miniaturization of conventional analytical equipment offers many advantages, such as short analysis time, low consumption of reactants, ease of automation, and integration of separation and detection on the same chip [40,41]. To utilize the full potential of miniaturized analytical instruments, there are strict requirements on the detectors, such as high sensitivity through low 438
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Figure 7. (a) Schematic diagram of the bonded glass chip, (b) channel layout, (c) photo of the device after drilling the holes and gluing the reservoirs. 1, Sample reservoir; 2, buffer reservoir; 3, sample waste; 4, buffer waste; 5, double-T injector; 6, separation channel; 7, floating platinum electrode; 8, sample filled into double-T injector; 9, vacuum; 10, direction of plug during separation. Adapted from [43].
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(a)
ITO electrode
unused reservoir buffer reservoir electrode plate
separation channel PMDS layer
sample reservoir
(b)
detection reservoir
ITO electrode
separation channel
The effective part (0.2 mm wide) containing with solution
Figure 8. Schematic of a microchip CE–ECL device: (a) top view of the PDMS layer and electrode plate; (b) an enlargement of the detection region. Adapted from [44].
working electrode and counter electrode. The voltage required to carry out the ECL reaction on the floating electrode was the result of the electric field presented in the separation channel during separation. Recently, our laboratory proposed an integrated indium tin oxide electrode-based Ru(bpy)32þ ECL detector for microchip CE device [44]. As illustrated in Fig. 8, this system utilized an ITO-coated glass slide as the chip substrate with a photolithographically fabricated ITO electrode located at the end of the separation channel. The separation and injection channels were contained in poly(dimethylsiloxane) (PDMS) layer, which was reversibly bound to the ITO electrode plate. With this construction, the alignment of the separation channel with the working electrode was greatly simplified and the photon-capturing efficiency enhanced [44].
7. Immobilization of the Ru(bpy)32 + on a solid surface The limitation of consuming expensive Ru(bpy)32þ can be overcome by immobilizing Ru(bpy)32þ in solid formats. Immobilization of Ru(bpy)32þ has several advantages over solution-phase ECL, such as simple experimental design and cost-effectiveness because there is no need to deliver extra Ru(bpy)32þ reagent. Directly synthesizing the polymer-containing Ru reagent at the surface is one mode of immobilization. Fresen et al. [45] developed a solid ECL sensor based on a poly[RuL3 ]2þ (where L ¼ vbpy or ester-substituted bipyridine) film on an array electrode or a sandwich-type arrangement. Light emission resulted from the reaction
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of RuL3þ and RuL33þ , which formed via independent control of the potentials of the closely spaced electrodes. Forster and Hogan [46] developed a new kind of electropolymerized [Ru(PVP-bpy)3 ]2þ -modified solid ECL electrode. But their analytical application was limited by the inhibition of mass transport within the polymer formed [45,46]. As a cation, Ru(bpy)32þ can be immobilized on some ion-exchange materials. Lin et al. [47] directly immobilized the Ru(bpy)32þ on a cationic ion-exchange resin and used it to determine oxalate, ethanol, and sulfite. The incorporation of Ru(bpy)32þ within the cationexchange polymer Nafion to fabricate an ECL-based sensor is still needed to improve reactivity and long-term stability [48]. Khramov and Collinson [49] reported the ECL of Ru(bpy)32þ ion-exchanged in Nafion–silica composite films, where the relatively slow diffusion of electroactive cations into Nafion can be improved by using Nafion–silica nanocomposites. We also reported the immobilization of Ru(bpy)32þ in the less hydrophobic PSS–silica (where PSS ¼ poly (sodium 4-styrene sulfonate) composite films [37,50,51], which were used as a solid ECL detector of CE [37] or electrostatically attached to the benzene sulfonic acid monolayer film [52]. Those ECL sensors showed good storage stability for six months. Eastman-AQ polymers are similar to Nafion in that they also contain sulfonated cation-exchange sites, but they are more hydrophilic than Nafion. Moreover, with their low cost, strong adherence to the surface, rapid response as well as permselective, ion-exchanging and anti-fouling properties, Eastman-AQ polymers appear to be attractive materials for sensor development. We incorporated AQ55D into silica sols and the resultant composite materials were used to immobilize Ru(bpy)32þ [53]. Compared with PSS, AQ55D does not need surfactant to prevent cracking of the composite film [53]. Choi et al. [28] reported an alternative matrix of composite films comprising sol–gel-derived titania and Nafion for the immobilization of Ru(bpy)32þ on an electrode surface and used it in HPLC detection. Modifying suitable material with Ru(bpy)32þ reagent has been used for the immobilization of Ru(bpy)32þ . Zhao et al. [54] reported on modified Ru(bpy)32þ chitosan coating on the Pt electrode. With this method, the response to trimethylamine was 39 times as weak as that obtained with a bare Pt electrode in an aqueous solution where Ru(bpy)32þ coexisted because of the electrostatic repulsion of the chitosan membrane having a positive charge to trimethylamine [54]. Compared with those methods, SiO2 -based gel is an attractive material because of its good three-dimension physical structure and high chemical stability, which allow the analyte to diffuse freely in the sol–gel film; but Ru(bpy)32þ leaching from the sol–gel film is the main drawback of this method [55]. It http://www.elsevier.com/locate/trac
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was also found that different co-reactants had different response times as a result of their different molecular structures, thus the diffusion of co-reactants differed [55]. Layer-by-layer (LBL) is an effective, attractive method often used for thin-film construction of polymers with oppositely charged colloidal particles. Dennany et al. [56] demonstrated direct ECL involving DNA in 10-nm films of cationic polymer [Ru(bpy)2 (PVP)10 ]2þ -assembled LBL with DNA. We have also used the simple, universal LBL technique to immobilize Ru(bpy)32þ [57]. Pre-made SiO2 nanoparticles, charged negatively at suitable pH, and Ru(bpy)32þ charged positively were used to form an ultrathin multilayer film via the LBL processing scheme, which was based on electrostatic interaction between positively charged Ru(bpy)32þ and negatively charged silica nanoparticles. After the [SiO2 /Ru(bpy)32þ ]n multilayer film was modified on ITO electrodes, we achieved high electroactivity, high stability and high reproducibility because of the high surface activity of the nanoparticles present [57]. 8. Conclusions ECL based on Ru(bpy)32þ has been successfully exploited as a sensitive detection technique for the determination of oxalate and a variety of amine-containing compounds in flowing streams, such as FIA, HPLC, CE and lTAS. Since Ru(bpy)32þ ECL emission is generated in situ in the reaction/detection cell with the original Ru(bpy)32þ species regenerated during the reaction, it does not need an external excitation light source and additional instrumentation to cut out scattered light for the excitation source, so sample dilution and band broadening are eliminated in a simple instrumental set-up. More applications of Ru(bpy)32þ ECL are required so that its inherent characteristics can be further exploited. We used Ru(bpy)32þ ECL to study the perchloratetriggered ion-gate behavior of a supported lipid bilayer membrane, which showed ion-gate behavior for the permeation of Ru(bpy)32þ in the presence of the perchlorate anion [58]. Zhan et al. [59,60] have devised a two-channel microfluidic sensor using anodic ECL as a photonic detector of cathodic redox reactions. The dual-electrode sensor relied on electrochemical detection at one electrode and ECL detection at the other. This approach allows the ECL reaction to be physically and chemically decoupled from the sensing channel of the device, which greatly expands the numbers of analytes that can be detected without derivatization. Acknowledgements This work is supported by the National Natural Science Foundation of China with Grant Nos. 20299030 and 440
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