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Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
C H A P T E R
32 Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection Estefanía Núñez-Bajo1, M. Teresa Fernández Abedul2 1
Department of Bioengineering, Royal School of Mines, Imperial College London, London, United Kingdom; 2Departamento de Química Física y Analítica, Universidad de Oviedo, Oviedo, Spain
32.1 Background Electrogenerated chemiluminescence or electrochemiluminescence (ECL) is a phenomenon in which the emission of photons is produced by a high-energy electron transfer reaction between electrogenerated species that is normally accompanied by the regeneration of the emitting species. Although the luminescence generated under electric fields was initially observed in the 1920s [1], it was reported in detail in the 1960s [2]. Since then, this detection technique has been widely studied and applied in many different fields (biosensors, microfluidics, etc.), as can be seen in the many articles that can be found in the literature. The mechanisms involved in ECL reactions depend on the emitting compound and the species that accompany it. Among others, the ECL of ruthenium complexes and their coreactants has been the most detailed and used in sensors and electron transfer studies [3]. Although modern ECL applications are based exclusively on the use of coreactants, the studies were originally based on ionic ECL annihilation. This mechanism involves the formation of an excited state as a result of an exergonic electron transfer (a process in which a reaction is followed by the emission of photons) between electrochemically generated species. As indicated below, after the emitter R is electrochemically oxidized (Eq. 32.1) and reduced (Eq. 32.2), the cationic (Rþ ) and anionic (R ) radicals are annihilated (Eq. 32.3) forming the excited species R , which emits photons when deactivated (Eq. 32.4).
Laboratory Methods in Dynamic Electroanalysis https://doi.org/10.1016/B978-0-12-815932-3.00032-2
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Copyright © 2020 Elsevier Inc. All rights reserved.
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
R e/Rþ R þ e/ R
ðoxidation on the electrodeÞ ðreduction on the electrodeÞ
Rþ þ R /R þ R R / R þ hn
ðannhilationÞ
ðlight emissionÞ
(32.1) (32.2) (32.3) (32.4)
When the annihilation reaction is not effective, the use of coreactants increases the intensity of the ECL signal. Unlike the annihilation of electrochemiluminescent ions, in which the electrolytic generation of the oxidized and reduced ECL precursors is required, the ECL with coreactant is usually generated by applying a unidirectional sweep of potentials in a solution containing luminophore species in the presence of an additional agent (coreactant). The redox intermediates of the coreactant are decomposed to produce a very reducing or oxidizing species. Because species with high oxidizing or reducing capacity are formed, ECL reactions are often referred to as “oxidative reduction” ECL (if the intermediate is a reducing agent generated by the electrochemical oxidation of the coreactant) and “reductive oxidation” ECL (if the intermediate is an oxidizing agent, product of an electrochemical reduction). Thus, a coreactant is a species that, after electrochemical oxidation or reduction, immediately undergoes chemical decomposition to form a strongly reducing or oxidizing intermediate. This can react with the oxidized or reduced luminophore, respectively, to generate excited states [4]. Then, the coreactant is consumed but, after ECL, the luminophore is regenerated. A good coreactant must meet the following conditions, it has: (i) to be soluble in the reaction medium since the ECL intensity is generally proportional to the coreactant concentration, (ii) to produce stable intermediate species in the solution under the electrolytic conditions, (iii) to be easily reduced or oxidized to quickly produce the chemical reaction that leads to the emission, and (iv) to not produce ECL extinction effects or be a luminophore species. Although there is a wide variety of molecules that exhibit ECL, most publications related to the use of coreactants involve the use of ruthenium organometallic complexes [5] because of their excellent chemical, electrochemical, and photochemical properties even in aqueous media and in the presence of oxygen [6]. In this chapter, [Ru(phen)3]2þ (luminophore) is used in combination with a tertiary amine (coreactant) where the mechanism generally consists of three basic steps: (i) redox reactions at the electrode, (ii) homogeneous chemical reactions, and (iii) formation of excited species. As the mechanisms related to the luminophore/coreactant pair are complex, and the main objective of the experiments is the familiarization with the analytical technique, a detailed description of the mechanism will be obviated. In this chapter, the dependence of the ECL signal on the luminophore and the medium is studied by cyclic voltammetry (CV) in [Ru(phen)3]2þ solutions. Once the electrochemical
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32.1 Background
and electrochemiluminescent behavior is characterized, the ECL generated by multipulsed amperometric detection (MAD) is evaluated as an alternative to CV. This technique has several advantages: (i) as a pulsed technique, it can increase the sensitivity because the capacitive current decays faster than the faradaic one, increasing the ratio between them (as ECL and electrochemical signals are related, this increase in sensitivity could have a direct effect on the ECL signal), (ii) in case monitorization of the signal with time is desired, application of pulses can be programmed for a long time without performing a manual control, and (iii) once the measurement is finished, file saving and loading is faster because a lower volume of data is obtained when compared with those coming from CV. Currently, all the commercially available analytical instruments are based on the ECL technology with the use of coreactant in aqueous media. The format and materials of the ECL instrumentation depend on the medium (organic or aqueous), the analyte, the introduction of measurement solutions (in flow or static configurations), and the scope of application (routine or decentralized analysis). Various commercial equipments can be found in the literature [7,8], although numerous studies are carried out in homemade instruments [9]. For the purposes highlighted above, a bipotentiostat/galvanostat and a UV-VIS spectrophotometer combined in a single commercial instrument (mStat-ECL) is used and controlled using specific software [10]. The electrochemiluminescent and electrochemical signals are simultaneously generated and captured (Fig. 32.1), which allows a better understanding of the electrochemical reactions involved in the light emission and the optimization of new analytical methodologies. The experiment is directed to graduate or Master students of courses related to the development of analytical methodologies (Chemistry, Biotechnology, etc.). It will give them, during a 6-h laboratory session (or two 3-h sessions), an appreciation of the basis of the electrochemiluminescent reactions and the optimization of the electrochemical parameters required as excitation source. They will know about a technique that includes both electrochemical and optical phenomena and is among the most sensitive for bioassays detection. Actually, it is included in many commercial autoanalyzers for high-throughput analysis.
Photodiode SPCE Connecon to the potenostat Lid with magnets SPCE
FIGURE 32.1 Different views of the electrochemiluminescence (ECL) cell that is connected to the potentiostat, controlled by a specific software that allows recording simultaneously electrochemical and ECL data. SPCE, screenprinted carbon electrode.
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
32.2 Chemicals and supplies Solutions
10 mM phosphate buffer (PB) pH 8.0. 10 mM Tris-HCl buffer (Tris-HCl) pH 8.0. 10 mM Tris-1 mM ethylenediaminetetraacetic acid (EDTA) buffer (TE) pH 8.0. Dichlorotris(1,10-phenanthroline) ruthenium(II) solution. A 20 mM stock solution of this compound should be prepared in 10 mM Tris-HCl pH 8.0. Milli-Q purified water is employed to prepare all buffer solutions. Instrumentation and materials mStat ECL bipotentiostat/galvanostat combined with the ECL cell (Si-photodiode integrated with spectral response range: 340e1100 nm), CAST connector, USB connector, and screen-printed carbon electrodes (SPCEs, DRP-110). All from Metrohm DropSens. Micropipettes (1e10, 10e100, and 100e1000 mL) with corresponding tips. Microcentrifuge tubes (0.5e1.5 mL), volumetric flasks (10, 25 mL), and other glassware material (beakers, etc.). Analytical weighing scale, pH meter, and laboratory spatulas.
32.3 Hazards EDTA causes serious eye irritation; hence, safety goggles have to be used in every moment. In case of eye irritation, eyes have to be rinsed cautiously with water for several minutes. The chemical, physical, and toxicological properties of dichlorotris (1,10-phenanthroline) ruthenium (II) have not been thoroughly investigated. It is not considered a hazardous substance according to Regulation (EC) No. 1272/2008 and is not classified as dangerous according to Directive 67/548/EEC. Students are required to wear gloves, eye protection, lab coat, and other appropriate protective equipment during this experiment.
32.4 Experimental procedure 32.4.1 General setup of the equipment 1. Assure that all the components (potentiostat and ECL cell) are correctly connected as shown in Fig. 32.1. 2. Connect a screen-printed card (SPCE) to the bipotentiostat/galvanostat instrument using the CAST connector. 3. Turn on the mSTAT-ECL instrument by pressing the button in the potentiostat, open the software, and connect the instrument. 4. Open the ECL cell, take out the lid with magnets, and place the SPCE on the slot of the base.
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32.4 Experimental procedure
5. Add 10 mL of solution over the three electrodes. 6. Place the lid with magnets carefully, avoiding the splash of the drop and fitting the O-ring on the top of the electrochemical cell (the working electrode should be the only electrode visible through the hole in the lid). 7. Add 40 mL of solution on the top of the electrodes through the hole of the lid with magnets. 8. Close the ECL cell and check that the Si-photodiode fits perfectly. 9. Perform dual electrochemical (EC)/ECL detection. 10. Choose the electrochemical technique (excitation source of ECL) and introduce the corresponding parameters. 11. Run the experiment, record the corresponding curve, and save it following the manual of the software. 12. Take out the clamp, disconnect the CAST connector, and open the ECL cell carefully. 13. Between experiments, take out the lid with magnets, rinse it with water, and dry it. 14. At the end, turn off the equipment, first the software and then the bipotentiostat/ galvanostat. In this chapter, CV and MAD are the methods applied for the electrochemical excitation. The parameters required for each experiment are commented in the corresponding section.
32.4.2 EC and ECL characterization of [Ru(Phen)3]2D by CV
ECL Intensity (counts)
Current Intensity ( A)
1. In a 500-mL microcentrifuge tube, prepare a 5 mM [Ru(Phen)3]2þ solution in Tris-HCl buffer pH 8.0 and follow the steps 1e10 described in Section 32.4.1, selecting CV as technique. 2. Introduce the following parameters: Estep ¼ 0.002 V, v ¼ 0.1 V/s, Ei ¼ þ0.3 V, E l ¼ þ1.4 V (Evtx1 in the software), Ef ¼ þ0.3 V (Evtx2 in the software). 3. Run the experiment and save the voltammogram (I vs. E curve) and ECL emission profile (IECL vs. E curve). An example is shown in Fig. 32.2.
Potential (V)
FIGURE 32.2 Cyclic voltammogram (blue) (dark gray in print version) and electrochemiluminescence (ECL) intensity (green) (gray in print version) versus potential profile recorded simultaneously in a 5 mM [Ru(phen)3]2þ solution in 10 mM Tris-HCl buffer pH 8.0.
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
4. Repeat the steps 1e3 of this section using two new electrodes. 5. Represent the ECL intensities and current intensities recorded versus the potential. 6. Discuss the redox processes involved. Indicate the average peak current intensity, maximum ECL intensity, and peak potentials.
32.4.3 Effect of the buffer composition on the ECL signal obtained by CV 1. Repeat the procedure described in Section 32.4.2 in different buffer solutions: PB solution, Tris-HCl, and TE pH 8.0 without [Ru(Phen)3]2þ and using one SPCE for each solution. No ECL signal should be observed. If there is any, the solution or the SPCE can contain impurities. Repeat after ensuring everything is clean. 2. In different microcentrifuge tubes of 500 mL, prepare the following solutions: 1 mM [Ru(Phen)3]2þ solution in 10 mM PB pH 8.0 1 mM [Ru(Phen)3]2þ solution in 10 mM Tris-HCl buffer pH 8.0 1 mM [Ru(Phen)3]2þ solution in 10 mM Tris-1mM EDTA (TE) buffer pH 8.0 3. Repeat the procedure described in Section 32.4.2 using three SPCEs for each solution (nine measurements). 4. Represent the ECL emission profiles (IECL vs. E) recorded in the different ruthenium solutions in a single graph by overlapping the most representative of each solution. Indicate the average ECL peak intensity and peak potential. 5. Considering the introduction section, explain the effect of the buffer on the ECL signal recorded in 1 mM [Ru(Phen)3]2þ solutions observing the voltammograms recorded in different buffer solutions. Choose the appropriate medium for the rest of experiments, as the one that provides the most intense ECL signal. 6. Discuss the excitation potential to be applied to perform MAD.
32.4.4 Optimization of the multipulsed amperometric detection 1. In a 1.5-mL microcentrifuge tube, prepare a 100 mM [Ru(Phen)3]2þ solution in 10 mM TE buffer pH 8.0. 2. Follow the steps 1e10 described in Section 32.4.1 selecting MAD as technique. The MAD procedure consists of three steps: in the first one the initial potential (E1) is maintained for a fixed time (t1), in a second one a pulse is applied, moving the potential to E2 for a time t2, and in the final one the potential is moved again to an E3 value (that can be equal to E1), where it is maintained for a t3 time. Then, the cycle is relaxation (t1 at E1)/excitation (t2 at E2)/relaxation (t3 at E3). In the case E3 is equal to E1, the procedure can be reduced to two steps: relaxation and excitation. Then, the potentials applied during the excitation and relaxation, as well as the time of application should be optimized. This is made in a one-factor-ata-time optimization, but an experimental design (see Chapter 35) could also be employed. 32.4.4.1 Optimization of the excitation pulse potential (E2) 1. Introduce the following parameters in the software: Number of steps: 3, interval: 0.1 s, repetitions: 2, cell 1: 1, E1: þ0.3 V, t1: 0.1 s, cell 2: 1, E2: þ1.4 V, t2: 0.5 s, cell 3: 1, E3: þ0.3 V and t3: 0.5 s.
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32.4 Experimental procedure
2. Run the experiment in a 100-mM [Ru(Phen)3]2þ solution in 10 mM TE buffer pH 8.0 and save the recorded ECL emission profiles (IECL vs. time). 3. Perform the same experiment applying þ1.3, þ1.35, and þ1.45 V of excitation pulse potential, washing the electrode between experiments. 4. Represent the ECL emission profiles (IECL vs. time) in a single graph and discuss the effect of the relaxation pulse potential on the ECL intensity. Choose the optimal potential for further experiments. 32.4.4.2 Optimization of the excitation pulse width (t2) 1. Introduce the following parameters in the software: Number of steps: 3, interval: 0.1 s, repetitions: 2, cell 1: 1, E1: þ0.3 V, t1: 0.1 s, cell 2: 1, E2: þ1.4 V, t2: 0.5 s, cell 3: 1, E3: þ0.3 V and t3: 0.5 s. 2. Run the experiment and save the recorded ECL emission profiles (IECL vs. time). An example is shown in Fig. 32.3. Perform the same experiment applying 0.1, 0.2, 1.0, 5.0, and 10 s as excitation pulse width (t2) washing the electrode between experiments. 3. Represent the electrochemical excitation signal (potential vs. time) indicating the excitation and relaxation zones in the scheme. 4. Represent the ECL emission profiles in a single graph and discuss the effect of the excitation pulse width on the maximum ECL intensity. 32.4.4.3 Optimization of the relaxation pulse potential (E1 and E3) 1. Introduce the following parameters in the software: Number of steps: 3, interval: 0.1 s, repetitions: 2, cell 1: 1, E1: þ0.3 V, t1: 0.1 s, cell 2: 1, E2: þ1.4 V, t2: 0.5 s, cell 3: 1, E3: þ0.3 V and t3: 0.5 s. 2. Run the experiment in 100 mM [Ru(Phen)3]2þ solution in 10 mM TE buffer pH 8.0 and save the recorded ECL emission profiles (IECL vs. time). 3. Perform the same experiment applying þ0.0, þ0.1, þ0.5, þ0.8, and þ1.0 V of relaxation pulse potential (E and E, where E ¼ E), washing the electrode between experiments.
Relaxation pulse
Emission pulse
Relaxation pulse
ECL Intensity (counts)
Emission pulse
Time (s)
FIGURE 32.3 Electrochemiluminescence (ECL) emission profile recorded in a 100 mM [Ru(phen)3]2þ solution in 10 mM TE buffer pH 8.0. Multipulsed amperometric detection (MAD) signals were recorded applying a MAD program (two repetitions).
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
4. Represent the ECL emission profiles (IECL vs. time) in a single graph and discuss the effect of the relaxation pulse potential on the ECL intensity. Choose the optimal potential for further experiments. 32.4.4.4 Optimization of the relaxation pulse width (t1 and t3) 1. Introduce the following parameters: Number of steps: 3, interval: 0.1 s, repetitions: 2, cell 1: 1, E1: potential chosen in the previous section, t1: 0.1 s, cell 2: 1, E2: þ1.4 V, t2: 0.5 s, cell 3: 1, E3: potential chosen in the previous section, t3: 0.5 s. 2. Run the experiment in a 100-mM [Ru(Phen)3]2þ solution in TE buffer pH 8.0 and save the ECL emission profile (IECL vs. time). 3. Perform the same experiment applying 0.1, 1, 5, and 10 s of relaxation pulse width (changing only t3). 4. Represent the ECL emission profiles in a single graph and discuss the effect of the relaxation pulse width on the ECL intensity.
32.4.5 Monitoring ECL emission (with multipulsed amperometric detection) with time
ECL Intensity (counts)
1. Considering the results obtained in Section 32.4.4, design a MAD program able to monitor the signal with time (e.g., for 20 min) in a 100-mM [Ru(Phen)3]2þ solution in TE buffer pH 8.0. Introduce the appropriate number of repetitions of a three-step program where the optimized excitation and relaxation parameters (E1, E2, E3, t1, t2, t3) are applied. 2. Run the experiment and save the recorded ECL emission profile (IECL vs. time). An example is shown in Fig. 32.4. 3. Represent the excitation signal (potential vs. time) and the recorded ECL emission profile (IECL vs. time). Discuss the stability of the signal with time, the advantages, and a possible analytical application of the method.
Time (s) Electrochemiluminescence (ECL) emission profile recorded in a 100-mM [Ru(phen)3]2þ solution in 10 mM TE buffer pH 8.0, following a multipulsed amperometric detection technology (nine repetitions).
FIGURE 32.4
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32.6 Additional notes
337
32.5 Lab report Write a lab report that includes an abstract, a brief introduction explaining the purpose of the experiment, a detailed experimental section, results and discussion, and conclusions. Include tables, graphics, or figures wherever necessary. The following points should be considered: 1. In the introduction, include a revision of reported works with examples of other electrochemiluminescent species and coreactants. Include also the most common ECL assays where ruthenium complexes are employed and a schematic representation of the components of the equipment. Remark the advantages and drawbacks of ECL with respect to chemiluminescence (CL) and other optical techniques. Indicate also the advantages of the combination of electrochemical and optical principles. 2. In the experimental section, explain the protocols to prepare the solutions, the equipment used, and the electrochemical techniques and parameters employed. Add schematic representations of the excitation signal (potential vs. time). 3. In the results and discussion section, show the most representative curves, including the incidences during the course of the experiment. Add a table comparing the results obtained during the optimization of the excitation and relaxation MAD parameters. Include a figure of the ECL signal recorded by MAD for 20 min and a schematic representation of the electrochemical excitation. 4. In the conclusions, highlight the most representative results and include possible applications of the MAD performed for recording measurements with time (e.g., 20 min).
32.6 Additional notes 1. All reagents in these experiments should be stored protected from light at 4 C. 2. Do not reuse SPCEs for concentrations higher than 1 mM of complex because of the possible poisoning of the electrode. 3. Dirt or strains on the window of the photodiode may cause a drop in light transmittance. Avoid the use of solvents as much as possible for cleaning the photodiode. If such use is unavoidable, use ethanol. Avoid touching the photodiode with bare hands and always use gloves. 4. The most intense signal should be obtained in TE buffer pH 8.0. This could be used to explain the effect of EDTA on the signal. 5. This chapter is thought for two sessions of 3 hours, but if the working time wants to be extended, the effect of the EDTA concentration and pH on the ECL signal could be studied. In addition, MAD experiments can be carried out in solutions with different concentrations of ruthenium complex to represent the calibration plot (IECL vs. concentration) and know the analytical characteristics of the method. Similarly, comparison with the EC and ECL behaviors of different ruthenium complexes (e.g., [Ru(bpy)3]2þ) could be made.
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
32.7 Assessment and discussion questions 1. Compare the electrochemiluminescent technique with the chemiluminescent and discuss the advantages over other optical detection techniques. 2. Considering the theory explained in the introduction, discuss the effect of the buffer solution, comment the type of mechanism in each case, and explain the effect of EDTA in the signal. Find other examples of coreactants. 3. What are the electrochemical techniques employed here for excitation? Comment the basis of both, deciding critically which one is the best. 4. Considering the stability of the MAD signal with time, comment possible applications of the methodology. 5. A dual detection, optical and electrochemical, can be made. What are the advantages? 6. Find in the bibliography electroactive species, not luminophores, what can affect the ECL signal and explain why.
References [1] R.T. Dufford, D. Nightingale, L.W. Gaddum, Luminescence of Grignard compounds in electric and magnetic fields, and related electrical phenomena, J. Am. Chem. Soc. 49 (1927) 1858e1864. [2] D.M. Hercules, Chemiluminescence resulting from electrochemically generated species, Science 145 (1964) 808e809. [3] A.J. Bard, G.M. Whitesides, Luminescent metal chelate labels and means for detection, U.S. Patent 5 (238) (1993) 808. [4] H.S. White, A.J. Bard, Electrogenerated chemiluminescence. 41. Electrogenerated chemiluminescence and chem2iluminescence of the Ru(2,21-bpy)2þ 3 -S2O8 system in acetonitrile-water solutions, J. Am. Chem. Soc. 104 (1982) 6891e6895. [5] R.M. Wightman, S.P. Forry, R. Maus, D. Badocco, P. Pastore, Rate-determining step in the electrogenerated chemiluminescence from tertiary amines with Tris(2,20-bipyridyl)ruthenium(II), J. Phys. Chem. B 108 (2004) 19119e19125. [6] A. Juris, V. Balzani, F. Barigelletti, S. Campagna, P. Belser, A. von Zelewsky, Ru(II) polypyridine complexes: photophysics, photochemistry, eletrochemistry, and chemiluminescence, Coord. Chem. Rev. 84 (1988) 85e277. [7] Meso Scale Discovery: www.mesoscale.com. [8] Roche Diagnostics Corporation: www.roche.com. [9] F.R.F. Fan, A.J. Bard, Electrogenerated Chemiluminescence, CRC Press, Boca Ratón, 2004, ISBN 9780824753474. [10] M.M.P.S. Neves, P. Bobes-Limenes, A. Pérez-Junquera, M.B. González-García, D. Hernández-Santos, P. FanjulBolado, Miniaturized analytical instrumentation for electrochemiluminescence assays: a spectrometer and a photodiode-based device, Anal. Bioanal. Chem. 408 (2016) 7121e7127.
VII. Spectroelectrochemical techniques
32 Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection Estefanía Núñez-Bajo1, M. Teresa Fernández Abedul2 1
Department of Bioengineering, Royal School of Mines, Imperial College London, London, United Kingdom; 2Departamento de Química Física y Analítica, Universidad de Oviedo, Oviedo, Spain
32.1 Background Electrogenerated chemiluminescence or electrochemiluminescence (ECL) is a phenomenon in which the emission of photons is produced by a high-energy electron transfer reaction between electrogenerated species that is normally accompanied by the regeneration of the emitting species. Although the luminescence generated under electric fields was initially observed in the 1920s [1], it was reported in detail in the 1960s [2]. Since then, this detection technique has been widely studied and applied in many different fields (biosensors, microfluidics, etc.), as can be seen in the many articles that can be found in the literature. The mechanisms involved in ECL reactions depend on the emitting compound and the species that accompany it. Among others, the ECL of ruthenium complexes and their coreactants has been the most detailed and used in sensors and electron transfer studies [3]. Although modern ECL applications are based exclusively on the use of coreactants, the studies were originally based on ionic ECL annihilation. This mechanism involves the formation of an excited state as a result of an exergonic electron transfer (a process in which a reaction is followed by the emission of photons) between electrochemically generated species. As indicated below, after the emitter R is electrochemically oxidized (Eq. 32.1) and reduced (Eq. 32.2), the cationic (Rþ ) and anionic (R ) radicals are annihilated (Eq. 32.3) forming the excited species R , which emits photons when deactivated (Eq. 32.4).
Laboratory Methods in Dynamic Electroanalysis https://doi.org/10.1016/B978-0-12-815932-3.00032-2
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Copyright © 2020 Elsevier Inc. All rights reserved.
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
R e/Rþ R þ e/ R
ðoxidation on the electrodeÞ ðreduction on the electrodeÞ
Rþ þ R /R þ R R / R þ hn
ðannhilationÞ
ðlight emissionÞ
(32.1) (32.2) (32.3) (32.4)
When the annihilation reaction is not effective, the use of coreactants increases the intensity of the ECL signal. Unlike the annihilation of electrochemiluminescent ions, in which the electrolytic generation of the oxidized and reduced ECL precursors is required, the ECL with coreactant is usually generated by applying a unidirectional sweep of potentials in a solution containing luminophore species in the presence of an additional agent (coreactant). The redox intermediates of the coreactant are decomposed to produce a very reducing or oxidizing species. Because species with high oxidizing or reducing capacity are formed, ECL reactions are often referred to as “oxidative reduction” ECL (if the intermediate is a reducing agent generated by the electrochemical oxidation of the coreactant) and “reductive oxidation” ECL (if the intermediate is an oxidizing agent, product of an electrochemical reduction). Thus, a coreactant is a species that, after electrochemical oxidation or reduction, immediately undergoes chemical decomposition to form a strongly reducing or oxidizing intermediate. This can react with the oxidized or reduced luminophore, respectively, to generate excited states [4]. Then, the coreactant is consumed but, after ECL, the luminophore is regenerated. A good coreactant must meet the following conditions, it has: (i) to be soluble in the reaction medium since the ECL intensity is generally proportional to the coreactant concentration, (ii) to produce stable intermediate species in the solution under the electrolytic conditions, (iii) to be easily reduced or oxidized to quickly produce the chemical reaction that leads to the emission, and (iv) to not produce ECL extinction effects or be a luminophore species. Although there is a wide variety of molecules that exhibit ECL, most publications related to the use of coreactants involve the use of ruthenium organometallic complexes [5] because of their excellent chemical, electrochemical, and photochemical properties even in aqueous media and in the presence of oxygen [6]. In this chapter, [Ru(phen)3]2þ (luminophore) is used in combination with a tertiary amine (coreactant) where the mechanism generally consists of three basic steps: (i) redox reactions at the electrode, (ii) homogeneous chemical reactions, and (iii) formation of excited species. As the mechanisms related to the luminophore/coreactant pair are complex, and the main objective of the experiments is the familiarization with the analytical technique, a detailed description of the mechanism will be obviated. In this chapter, the dependence of the ECL signal on the luminophore and the medium is studied by cyclic voltammetry (CV) in [Ru(phen)3]2þ solutions. Once the electrochemical
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32.1 Background
and electrochemiluminescent behavior is characterized, the ECL generated by multipulsed amperometric detection (MAD) is evaluated as an alternative to CV. This technique has several advantages: (i) as a pulsed technique, it can increase the sensitivity because the capacitive current decays faster than the faradaic one, increasing the ratio between them (as ECL and electrochemical signals are related, this increase in sensitivity could have a direct effect on the ECL signal), (ii) in case monitorization of the signal with time is desired, application of pulses can be programmed for a long time without performing a manual control, and (iii) once the measurement is finished, file saving and loading is faster because a lower volume of data is obtained when compared with those coming from CV. Currently, all the commercially available analytical instruments are based on the ECL technology with the use of coreactant in aqueous media. The format and materials of the ECL instrumentation depend on the medium (organic or aqueous), the analyte, the introduction of measurement solutions (in flow or static configurations), and the scope of application (routine or decentralized analysis). Various commercial equipments can be found in the literature [7,8], although numerous studies are carried out in homemade instruments [9]. For the purposes highlighted above, a bipotentiostat/galvanostat and a UV-VIS spectrophotometer combined in a single commercial instrument (mStat-ECL) is used and controlled using specific software [10]. The electrochemiluminescent and electrochemical signals are simultaneously generated and captured (Fig. 32.1), which allows a better understanding of the electrochemical reactions involved in the light emission and the optimization of new analytical methodologies. The experiment is directed to graduate or Master students of courses related to the development of analytical methodologies (Chemistry, Biotechnology, etc.). It will give them, during a 6-h laboratory session (or two 3-h sessions), an appreciation of the basis of the electrochemiluminescent reactions and the optimization of the electrochemical parameters required as excitation source. They will know about a technique that includes both electrochemical and optical phenomena and is among the most sensitive for bioassays detection. Actually, it is included in many commercial autoanalyzers for high-throughput analysis.
Photodiode SPCE Connecon to the potenostat Lid with magnets SPCE
FIGURE 32.1 Different views of the electrochemiluminescence (ECL) cell that is connected to the potentiostat, controlled by a specific software that allows recording simultaneously electrochemical and ECL data. SPCE, screenprinted carbon electrode.
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
32.2 Chemicals and supplies Solutions
10 mM phosphate buffer (PB) pH 8.0. 10 mM Tris-HCl buffer (Tris-HCl) pH 8.0. 10 mM Tris-1 mM ethylenediaminetetraacetic acid (EDTA) buffer (TE) pH 8.0. Dichlorotris(1,10-phenanthroline) ruthenium(II) solution. A 20 mM stock solution of this compound should be prepared in 10 mM Tris-HCl pH 8.0. Milli-Q purified water is employed to prepare all buffer solutions. Instrumentation and materials mStat ECL bipotentiostat/galvanostat combined with the ECL cell (Si-photodiode integrated with spectral response range: 340e1100 nm), CAST connector, USB connector, and screen-printed carbon electrodes (SPCEs, DRP-110). All from Metrohm DropSens. Micropipettes (1e10, 10e100, and 100e1000 mL) with corresponding tips. Microcentrifuge tubes (0.5e1.5 mL), volumetric flasks (10, 25 mL), and other glassware material (beakers, etc.). Analytical weighing scale, pH meter, and laboratory spatulas.
32.3 Hazards EDTA causes serious eye irritation; hence, safety goggles have to be used in every moment. In case of eye irritation, eyes have to be rinsed cautiously with water for several minutes. The chemical, physical, and toxicological properties of dichlorotris (1,10-phenanthroline) ruthenium (II) have not been thoroughly investigated. It is not considered a hazardous substance according to Regulation (EC) No. 1272/2008 and is not classified as dangerous according to Directive 67/548/EEC. Students are required to wear gloves, eye protection, lab coat, and other appropriate protective equipment during this experiment.
32.4 Experimental procedure 32.4.1 General setup of the equipment 1. Assure that all the components (potentiostat and ECL cell) are correctly connected as shown in Fig. 32.1. 2. Connect a screen-printed card (SPCE) to the bipotentiostat/galvanostat instrument using the CAST connector. 3. Turn on the mSTAT-ECL instrument by pressing the button in the potentiostat, open the software, and connect the instrument. 4. Open the ECL cell, take out the lid with magnets, and place the SPCE on the slot of the base.
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32.4 Experimental procedure
5. Add 10 mL of solution over the three electrodes. 6. Place the lid with magnets carefully, avoiding the splash of the drop and fitting the O-ring on the top of the electrochemical cell (the working electrode should be the only electrode visible through the hole in the lid). 7. Add 40 mL of solution on the top of the electrodes through the hole of the lid with magnets. 8. Close the ECL cell and check that the Si-photodiode fits perfectly. 9. Perform dual electrochemical (EC)/ECL detection. 10. Choose the electrochemical technique (excitation source of ECL) and introduce the corresponding parameters. 11. Run the experiment, record the corresponding curve, and save it following the manual of the software. 12. Take out the clamp, disconnect the CAST connector, and open the ECL cell carefully. 13. Between experiments, take out the lid with magnets, rinse it with water, and dry it. 14. At the end, turn off the equipment, first the software and then the bipotentiostat/ galvanostat. In this chapter, CV and MAD are the methods applied for the electrochemical excitation. The parameters required for each experiment are commented in the corresponding section.
32.4.2 EC and ECL characterization of [Ru(Phen)3]2D by CV
ECL Intensity (counts)
Current Intensity ( A)
1. In a 500-mL microcentrifuge tube, prepare a 5 mM [Ru(Phen)3]2þ solution in Tris-HCl buffer pH 8.0 and follow the steps 1e10 described in Section 32.4.1, selecting CV as technique. 2. Introduce the following parameters: Estep ¼ 0.002 V, v ¼ 0.1 V/s, Ei ¼ þ0.3 V, E l ¼ þ1.4 V (Evtx1 in the software), Ef ¼ þ0.3 V (Evtx2 in the software). 3. Run the experiment and save the voltammogram (I vs. E curve) and ECL emission profile (IECL vs. E curve). An example is shown in Fig. 32.2.
Potential (V)
FIGURE 32.2 Cyclic voltammogram (blue) (dark gray in print version) and electrochemiluminescence (ECL) intensity (green) (gray in print version) versus potential profile recorded simultaneously in a 5 mM [Ru(phen)3]2þ solution in 10 mM Tris-HCl buffer pH 8.0.
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
4. Repeat the steps 1e3 of this section using two new electrodes. 5. Represent the ECL intensities and current intensities recorded versus the potential. 6. Discuss the redox processes involved. Indicate the average peak current intensity, maximum ECL intensity, and peak potentials.
32.4.3 Effect of the buffer composition on the ECL signal obtained by CV 1. Repeat the procedure described in Section 32.4.2 in different buffer solutions: PB solution, Tris-HCl, and TE pH 8.0 without [Ru(Phen)3]2þ and using one SPCE for each solution. No ECL signal should be observed. If there is any, the solution or the SPCE can contain impurities. Repeat after ensuring everything is clean. 2. In different microcentrifuge tubes of 500 mL, prepare the following solutions: 1 mM [Ru(Phen)3]2þ solution in 10 mM PB pH 8.0 1 mM [Ru(Phen)3]2þ solution in 10 mM Tris-HCl buffer pH 8.0 1 mM [Ru(Phen)3]2þ solution in 10 mM Tris-1mM EDTA (TE) buffer pH 8.0 3. Repeat the procedure described in Section 32.4.2 using three SPCEs for each solution (nine measurements). 4. Represent the ECL emission profiles (IECL vs. E) recorded in the different ruthenium solutions in a single graph by overlapping the most representative of each solution. Indicate the average ECL peak intensity and peak potential. 5. Considering the introduction section, explain the effect of the buffer on the ECL signal recorded in 1 mM [Ru(Phen)3]2þ solutions observing the voltammograms recorded in different buffer solutions. Choose the appropriate medium for the rest of experiments, as the one that provides the most intense ECL signal. 6. Discuss the excitation potential to be applied to perform MAD.
32.4.4 Optimization of the multipulsed amperometric detection 1. In a 1.5-mL microcentrifuge tube, prepare a 100 mM [Ru(Phen)3]2þ solution in 10 mM TE buffer pH 8.0. 2. Follow the steps 1e10 described in Section 32.4.1 selecting MAD as technique. The MAD procedure consists of three steps: in the first one the initial potential (E1) is maintained for a fixed time (t1), in a second one a pulse is applied, moving the potential to E2 for a time t2, and in the final one the potential is moved again to an E3 value (that can be equal to E1), where it is maintained for a t3 time. Then, the cycle is relaxation (t1 at E1)/excitation (t2 at E2)/relaxation (t3 at E3). In the case E3 is equal to E1, the procedure can be reduced to two steps: relaxation and excitation. Then, the potentials applied during the excitation and relaxation, as well as the time of application should be optimized. This is made in a one-factor-ata-time optimization, but an experimental design (see Chapter 35) could also be employed. 32.4.4.1 Optimization of the excitation pulse potential (E2) 1. Introduce the following parameters in the software: Number of steps: 3, interval: 0.1 s, repetitions: 2, cell 1: 1, E1: þ0.3 V, t1: 0.1 s, cell 2: 1, E2: þ1.4 V, t2: 0.5 s, cell 3: 1, E3: þ0.3 V and t3: 0.5 s.
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32.4 Experimental procedure
2. Run the experiment in a 100-mM [Ru(Phen)3]2þ solution in 10 mM TE buffer pH 8.0 and save the recorded ECL emission profiles (IECL vs. time). 3. Perform the same experiment applying þ1.3, þ1.35, and þ1.45 V of excitation pulse potential, washing the electrode between experiments. 4. Represent the ECL emission profiles (IECL vs. time) in a single graph and discuss the effect of the relaxation pulse potential on the ECL intensity. Choose the optimal potential for further experiments. 32.4.4.2 Optimization of the excitation pulse width (t2) 1. Introduce the following parameters in the software: Number of steps: 3, interval: 0.1 s, repetitions: 2, cell 1: 1, E1: þ0.3 V, t1: 0.1 s, cell 2: 1, E2: þ1.4 V, t2: 0.5 s, cell 3: 1, E3: þ0.3 V and t3: 0.5 s. 2. Run the experiment and save the recorded ECL emission profiles (IECL vs. time). An example is shown in Fig. 32.3. Perform the same experiment applying 0.1, 0.2, 1.0, 5.0, and 10 s as excitation pulse width (t2) washing the electrode between experiments. 3. Represent the electrochemical excitation signal (potential vs. time) indicating the excitation and relaxation zones in the scheme. 4. Represent the ECL emission profiles in a single graph and discuss the effect of the excitation pulse width on the maximum ECL intensity. 32.4.4.3 Optimization of the relaxation pulse potential (E1 and E3) 1. Introduce the following parameters in the software: Number of steps: 3, interval: 0.1 s, repetitions: 2, cell 1: 1, E1: þ0.3 V, t1: 0.1 s, cell 2: 1, E2: þ1.4 V, t2: 0.5 s, cell 3: 1, E3: þ0.3 V and t3: 0.5 s. 2. Run the experiment in 100 mM [Ru(Phen)3]2þ solution in 10 mM TE buffer pH 8.0 and save the recorded ECL emission profiles (IECL vs. time). 3. Perform the same experiment applying þ0.0, þ0.1, þ0.5, þ0.8, and þ1.0 V of relaxation pulse potential (E and E, where E ¼ E), washing the electrode between experiments.
Relaxation pulse
Emission pulse
Relaxation pulse
ECL Intensity (counts)
Emission pulse
Time (s)
FIGURE 32.3 Electrochemiluminescence (ECL) emission profile recorded in a 100 mM [Ru(phen)3]2þ solution in 10 mM TE buffer pH 8.0. Multipulsed amperometric detection (MAD) signals were recorded applying a MAD program (two repetitions).
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
4. Represent the ECL emission profiles (IECL vs. time) in a single graph and discuss the effect of the relaxation pulse potential on the ECL intensity. Choose the optimal potential for further experiments. 32.4.4.4 Optimization of the relaxation pulse width (t1 and t3) 1. Introduce the following parameters: Number of steps: 3, interval: 0.1 s, repetitions: 2, cell 1: 1, E1: potential chosen in the previous section, t1: 0.1 s, cell 2: 1, E2: þ1.4 V, t2: 0.5 s, cell 3: 1, E3: potential chosen in the previous section, t3: 0.5 s. 2. Run the experiment in a 100-mM [Ru(Phen)3]2þ solution in TE buffer pH 8.0 and save the ECL emission profile (IECL vs. time). 3. Perform the same experiment applying 0.1, 1, 5, and 10 s of relaxation pulse width (changing only t3). 4. Represent the ECL emission profiles in a single graph and discuss the effect of the relaxation pulse width on the ECL intensity.
32.4.5 Monitoring ECL emission (with multipulsed amperometric detection) with time
ECL Intensity (counts)
1. Considering the results obtained in Section 32.4.4, design a MAD program able to monitor the signal with time (e.g., for 20 min) in a 100-mM [Ru(Phen)3]2þ solution in TE buffer pH 8.0. Introduce the appropriate number of repetitions of a three-step program where the optimized excitation and relaxation parameters (E1, E2, E3, t1, t2, t3) are applied. 2. Run the experiment and save the recorded ECL emission profile (IECL vs. time). An example is shown in Fig. 32.4. 3. Represent the excitation signal (potential vs. time) and the recorded ECL emission profile (IECL vs. time). Discuss the stability of the signal with time, the advantages, and a possible analytical application of the method.
Time (s) Electrochemiluminescence (ECL) emission profile recorded in a 100-mM [Ru(phen)3]2þ solution in 10 mM TE buffer pH 8.0, following a multipulsed amperometric detection technology (nine repetitions).
FIGURE 32.4
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32.6 Additional notes
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32.5 Lab report Write a lab report that includes an abstract, a brief introduction explaining the purpose of the experiment, a detailed experimental section, results and discussion, and conclusions. Include tables, graphics, or figures wherever necessary. The following points should be considered: 1. In the introduction, include a revision of reported works with examples of other electrochemiluminescent species and coreactants. Include also the most common ECL assays where ruthenium complexes are employed and a schematic representation of the components of the equipment. Remark the advantages and drawbacks of ECL with respect to chemiluminescence (CL) and other optical techniques. Indicate also the advantages of the combination of electrochemical and optical principles. 2. In the experimental section, explain the protocols to prepare the solutions, the equipment used, and the electrochemical techniques and parameters employed. Add schematic representations of the excitation signal (potential vs. time). 3. In the results and discussion section, show the most representative curves, including the incidences during the course of the experiment. Add a table comparing the results obtained during the optimization of the excitation and relaxation MAD parameters. Include a figure of the ECL signal recorded by MAD for 20 min and a schematic representation of the electrochemical excitation. 4. In the conclusions, highlight the most representative results and include possible applications of the MAD performed for recording measurements with time (e.g., 20 min).
32.6 Additional notes 1. All reagents in these experiments should be stored protected from light at 4 C. 2. Do not reuse SPCEs for concentrations higher than 1 mM of complex because of the possible poisoning of the electrode. 3. Dirt or strains on the window of the photodiode may cause a drop in light transmittance. Avoid the use of solvents as much as possible for cleaning the photodiode. If such use is unavoidable, use ethanol. Avoid touching the photodiode with bare hands and always use gloves. 4. The most intense signal should be obtained in TE buffer pH 8.0. This could be used to explain the effect of EDTA on the signal. 5. This chapter is thought for two sessions of 3 hours, but if the working time wants to be extended, the effect of the EDTA concentration and pH on the ECL signal could be studied. In addition, MAD experiments can be carried out in solutions with different concentrations of ruthenium complex to represent the calibration plot (IECL vs. concentration) and know the analytical characteristics of the method. Similarly, comparison with the EC and ECL behaviors of different ruthenium complexes (e.g., [Ru(bpy)3]2þ) could be made.
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32. Electrochemiluminescence of tris (1,10-phenanthroline) ruthenium(II) complex with multipulsed amperometric detection
32.7 Assessment and discussion questions 1. Compare the electrochemiluminescent technique with the chemiluminescent and discuss the advantages over other optical detection techniques. 2. Considering the theory explained in the introduction, discuss the effect of the buffer solution, comment the type of mechanism in each case, and explain the effect of EDTA in the signal. Find other examples of coreactants. 3. What are the electrochemical techniques employed here for excitation? Comment the basis of both, deciding critically which one is the best. 4. Considering the stability of the MAD signal with time, comment possible applications of the methodology. 5. A dual detection, optical and electrochemical, can be made. What are the advantages? 6. Find in the bibliography electroactive species, not luminophores, what can affect the ECL signal and explain why.
References [1] R.T. Dufford, D. Nightingale, L.W. Gaddum, Luminescence of Grignard compounds in electric and magnetic fields, and related electrical phenomena, J. Am. Chem. Soc. 49 (1927) 1858e1864. [2] D.M. Hercules, Chemiluminescence resulting from electrochemically generated species, Science 145 (1964) 808e809. [3] A.J. Bard, G.M. Whitesides, Luminescent metal chelate labels and means for detection, U.S. Patent 5 (238) (1993) 808. [4] H.S. White, A.J. Bard, Electrogenerated chemiluminescence. 41. Electrogenerated chemiluminescence and chem2iluminescence of the Ru(2,21-bpy)2þ 3 -S2O8 system in acetonitrile-water solutions, J. Am. Chem. Soc. 104 (1982) 6891e6895. [5] R.M. Wightman, S.P. Forry, R. Maus, D. Badocco, P. Pastore, Rate-determining step in the electrogenerated chemiluminescence from tertiary amines with Tris(2,20-bipyridyl)ruthenium(II), J. Phys. Chem. B 108 (2004) 19119e19125. [6] A. Juris, V. Balzani, F. Barigelletti, S. Campagna, P. Belser, A. von Zelewsky, Ru(II) polypyridine complexes: photophysics, photochemistry, eletrochemistry, and chemiluminescence, Coord. Chem. Rev. 84 (1988) 85e277. [7] Meso Scale Discovery: www.mesoscale.com. [8] Roche Diagnostics Corporation: www.roche.com. [9] F.R.F. Fan, A.J. Bard, Electrogenerated Chemiluminescence, CRC Press, Boca Ratón, 2004, ISBN 9780824753474. [10] M.M.P.S. Neves, P. Bobes-Limenes, A. Pérez-Junquera, M.B. González-García, D. Hernández-Santos, P. FanjulBolado, Miniaturized analytical instrumentation for electrochemiluminescence assays: a spectrometer and a photodiode-based device, Anal. Bioanal. Chem. 408 (2016) 7121e7127.
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