Spectroscopic Properties of Inorganic and Organometallic Compounds: Techniques, Materials and Applications, Volume 44

Spectroscopic Properties of Inorganic and Organometallic Compounds: Techniques, Materials and Applications, Volume 44


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Product Details

ISBN-13: 9781849735797
Publisher: Royal Society of Chemistry, The
Publication date: 07/31/2013
Series: Specialist Periodical Reports Series , #44
Pages: 155
Product dimensions: 6.14(w) x 9.21(h) x 0.56(d)

About the Author

Professor Jack Yarwood is an emeritus professor at Sheffield Hallam University. Professor Simon Duckett is a research group leader at the University of York, UK. His group is mainly involved in the design, development and implementation of NMR methods, supported by the synthesis of inorganic and organometallic complexes. Dr Richard Douthwaite is at the University of York, UK. His main research interests include molecular and materials chemistry and photocatalysis. Both an EPSRC college member and fellow of the Royal Society of Chemistry, Dr DOuthwaite is also on the SCI National Materials Committee.

Read an Excerpt

Spectroscopic Properties of Inorganic and Organometallic Compounds

Techniques, Materials and Applications Volume 44

By J. Yarwood, R. Douthwaite, S. B. Duckett

The Royal Society of Chemistry

Copyright © 2013 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-579-7


Electrochemiluminesence of ruthenium complex and its application in biosensors

Jing Li, Xiaofang Jia and Erkang Wang

DOI: 10.1039/9781849737791-00001

Electrochemiluminesence (ECL), also called electrogenerated chemiluminescence, is optical emission that arises from the high-energy electron-transfer reaction between electrogenerated species. ECL is an approach of converting electrical energy into radiative energy. Different from photoluminescence, ECL does not require the use of external light sources and therefore problems derived by light scattering can be avoided.

1 Introduction

Electrochemiluminesence (ECL), also called electrogenerated chemiluminescence, is optical emission that arises from the high-energy electron-transfer reaction between electrogenerated species. ECL is an approach of converting electrical energy into radiative energy. Different from photo-luminescence, ECL does not require the use of external light sources and therefore problems derived by light scattering can be avoided. As an important analytical method, ECL, a marriage of chemiluminescence (CL) and electrochemistry, exhibits potential advantages over CL: (1) ECL allows the time and position of the light-emitting reactions to be accurately controlled by applying a suitable potential on an electrode surface. By controlling time, ECL can be obtained until some reactions have taken place. The better control over the emission position by confining light emission to a region that is precisely located with respect to the detector can be beneficial for sensitivity by dramatically improving the signal-to-noise ratio. In addition, control over position enables the determination of multi-analytes by interrogating each electrode in an array with the development of ECL microscopy, (2) ECL can be initiated selectively by switching the electrode potentials, (3) Some ECL emitters can be regenerated during the ECL process, which greatly enhances the sensitivity of the technique, saves reactants and simplifies the set-up, (4) During the ECL process, the current signal and light signal are obtained simultaneously, which facilitate the investigation of light emission mechanism by electrochemical methods.

Since the first detailed investigations about the ECL emission were reported in the mid-1960s, a variety of ECL emitters have been explored including organic (e.g., luminol), inorganic (e.g., metal complexes) and nanomaterials based ECL systems (e.g., QDs). Among them, ECL of ruthenium complex and its co-reactants as a sensitive detection method have been extensively investigated, especially the application of Ru(bpy)32+.

Up to now, ECL assays based on the ruthenium complex have been elaborately designed and widely used in the areas of clinical diagnostics, food and water testing, environmental monitoring, biowarfare agent detection, and scientific research. Progress in the field were summarized in several excellent review articles. And certain types of ECL instruments are now commercially available, such as cobas e 411 analyzer from Roche Diagnostics Corp., Sector PR Reader 400 by Meso Scale Discovery Corporation and capillary electrophoresis (CE)-ECL system developed by the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, and manufactured by Xi'an Remax Electronic Co. LTD. The basic components of an ECL instrument include an electrical energy supply for initiating the ECL reaction at an electrode within an electrochemical cell and an optical detection system for measurement of either the emitted light intensity(for quantitative analysis) or its spectroscopic response (for qualitative analysis). The light detection system can be integrated by a photo- multiplier tube (PMT) biased at a high voltage with a high-voltage power supply, a charged coupled device (CCD) or a photodiode. The utilization of a PMT can provide the most sensitive way to detect light with single photons. The use of CCD camera has received more attention in ECL imaging and high throughput analysis owing to instant image manipulation, high spatial resolution, and multi-channel detection ability.

In this chapter, we summarize advances in the development of ruthenium complex based ECL biosensors mainly focusing on the principle of ECL and their applications in the fabrication of various biosensors.

2 Principle of ECL

There are two dominant pathways through which ECL can be obtained: annihilation pathway and co-reactant pathway. Although most of the ECL applications are based on the co-reactant pathway, the early ECL emission originated from annihilation ECL.

2.1 Annihilation ECL

In the annihilation pathway, the reduced and oxidized species are both generated in the vicinity of the electrode surface by alternate pulsing of the electrode potential. The corresponding process is outlined in the Eqn (1–4). The annihilation ECL can also be achieved in the "mixed systems". The ECL is achieved via "cross-reactions" between the radical ions of the different species.

R - e- -> R•+ (Oxidation at electrode) (1)

R + e- -> R•+ (Reduction at electrode) (2)

R•+ + R•- -> R* + R (Excited state formation) (3)

R* -> R + hv (Light emission) (4)

The excited state R* can represent the lowest singlet state species (1R*) or triplet state species (3R*) according to the energy available during the annihilation reaction. If the enthalpy of the ion annihilation (ΔH) exceeds the energy required to produce the lowest excited states from the ground state (Es, which can be obtained spectroscopically), the light emission process follows the singlet route "S-route" (Eqn (3a)) and the system is called the energy-sufficient system. ΔH can be calculated based on Eqn (5), where TΔS is estimated to be about 0.1 eV at 25 °C, E0 is reversible standard potentials of the redox couples. Most ECL systems based on aromatic compounds are in accordance with this mechanism, such as the ECL of rubrene (ΔH obtained from the electron-transfer reaction is 2.32 eV, and Es is 2.30 eV). Another example of an energy sufficient system is the inorganic species Ru(bpy)32+ (ΔH is ca. 2.6 eV, and Es lies at 2.10 eV with λmax = 620 nm).


(1) S-route

R•+ + R•- ->1R* + R (Excited singlet formation) (3a)

In contrast, if ΔH is smaller than Es but larger than the triplet state energy (Et), 3R* is initially formed, and then 1R* can be formed by the triplet- triplet annihilation (TTA). The proposed mechanism involves triplet intermediates and is defined as the "'T-route"'. Such system is called "energy deficient". A typical example of TTA annihilation is the ECL of DPA/TMPD (DPA=9,10-diphenylanthracene and TMPD=N,N,N',N'-tetra-methyl-p-phenylenediamine) system. ΔH of 2.03 eV is much less than the energy of 3.00 eV required to reach the emitting singlet excited state for DPA. The efficiency of direct emission from 3R* is usually low in a solution phase because the long radiative lifetime of 3R* resulted in its easy quenching by radical ions or other species, such as molecular oxygen. ECL reactions with different precursors follow this route.

(2) T-route

(2) R•+ + R•- ->3R* + R (Excited triplet formation) (3b)

(3) 3R* + 3R* ->1R* + R (TTA) (6)

In most cases, annihilation pathway based ECL is generated at a single electrode. It is possible to obtain emission with two differe electrodes that are close enough to allow the electrogenerated reactants to interdiffuse and undergo annihilation, such as the use of a rotating ring disk electrode and dual-working electrode with interdigitated electrodes. Annihilation path-way based ECL is widely used in light-emitting electrochemical cells.

For the efficient generation of annihilation ECL, three conditions should be met as follows: (1) sufficient energy in the electron transfer reaction to produce the excited state for ECL emission, (2) stable radical ions of the precursor molecules in the electrolyte, and (3) good PL efficiency of a product of the electron transfer reaction.

2.2 Co-reactant ECL

The co-reactant ECL is usually generated with the reaction between the luminophore species and an additionally added assistant reagent (co-reactant) by one directional potential scanning or a single potential step. The first co-reactant discovered was oxalate in 1977. The introduction of the co-reactant in ECL exhibits distinct advantage in comparison with the annihilation reaction: (1) it can overcome the limited potential window of solvent and the poor stability of radical anions or cations; (2) the co-reactant ECL can be beneficial for some fluorescent compounds that have only a electrochemical reduction or oxidation; (3) the use of co-reactant can produce more intense ECL emission when the annihilation reaction between oxidized and reduced species is not efficient; (4) it can eliminate the oxygen quenching effect frequently encountered in ion annihilation reaction and facilitate the ECL in the air. All commercially available ECL analytical instruments are based on this pathway. According to the generated intermediates and the polarity of the applied potential, the corresponding "co-reactant ECL" can be classified as "oxidative-reduction" ECL and "reductive-oxidation" ECL, respectively.

2.2.1 Oxidative-Reduction ECL. The ECL system involving the generation of a strong reducing intermediate species upon electrochemical oxidation was defined as "'oxidative-reduction" ECL. The ECL of Ru(bpy)32+/oxalate (C2O42-) system is a typical example. The corresponding mechanism was described in Eqn (7)–(13).

Ru(bpy)32+ - e- -> Ru{bpy)33+ (7)


C2O4•- -> CO2•- + CO2 (9)

Ru(bpy)32+ + CO2•- -> Ru(bpy)32+ + CO2 (10)

Ru(bpy)32+ + CO2•- -> Ru(bpy)3+ + CO2 (11)

Ru(bpy)33+ + Ru(bpy)3+ -> Ru(bpy)32+* + Ru(bpy)32+ (12)

Ru(bpy)32+* -> Ru(bpy)32+ + hv (13)

During this ECL process, a powerful reductant (CO2•-) was in situ generated due to the decomposition of intermediate (C2O4•-). The ECL can be achieved by two pathways: (1) reaction between the CO2•- and Ru(bpy)3+ (Eqn (10)), (2) ion annihilation reaction between Ru(bpy)3+ and Ru(bpy)33+ (Eqn (12)). The intermediate C2O4•- can be formed upon oxidation by Ru(bpy)33+ or be directly oxidized at the electrode surface (Eqn (8)). For example, in acetonitrile (MeCN) media, oxalate is easier to be oxidized than Ru(bpy)32+ complex and both the reactants are oxidized during the light emission. In addition, the applied potential, the concentration of C2O42- and the electrode surface properties influence the direct oxidation of oxalate to the overall ECL behaviour.

Another popular "oxidative-reduction" example is Ru(bpy)32+/tripropylamine (TPrA) system. It is the basis of commercial systems for immunoassay and DNA analysis due to the high ECL efficiency. The mechanism of Ru(bpy)32+/TPrA system is very complicated and has been elucidated in detail in the literature. Generally, the ECL emission spectra of this system as a function of applied potential generally consist of two waves (Fig. 1). The first ECL wave occurs at the direct oxidation potential of TPrA and largely depends on the concentration of the analytes. In diluted Ru(bpy)32+ solution (<μM) containing 0.1 M TPrA, the ECL intensity of first wave is often obvious.

The detailed reaction mechanism for the first wave is described in scheme 1, Fig. 2. Upon electrochemical oxidation, the cation radical species TprA•+ and free radical TprA• are generated. The following reaction between TprA•+ and Ru(bpy)32+ generates Ru(bpy)3+ and it reacts with TprA•+ to form the excited state Ru(bpy)32+*. This ECL process is defined as low oxidation potential (LOP) ECL, where the potential applied is positive enough for the oxidation of TPrA, but not enough for the oxidation of Ru(bpy)32+. The lower potentials required in the LOP ECL is favorable for DNA diagnostic applications because oligonucleotide sequences would be irreversibly damaged at potentials above + 1.0 V vs. SCE.

However, for the second ECL wave, it is closely related to the oxidation of Ru(bpy)32+ at the electrode surface (scheme 2-4). Ru(bpy)32+ is firstly oxidized electrochemically to form Ru(bpy)33+ and the strong reductant TprA• is formed either through the direct oxidation of TPrA at the electrode surface (scheme 2 and 3) or homogeneous oxidation of TPrA with Ru(bpy)33+ (scheme 4). And then the excited state Ru(bpy)32+* forms through three different reactions: (1) ion annihilation reaction between Ru(bpy)33+ and Ru(bpy)3+ [generated from the reduction of Ru(bpy)32+ by TprA• free radical] (scheme 3), (2) Ru(bpy)3+ oxidation with cation radical species TprA•+ as the same as the first ECL wave (scheme 1), (3) Ru(bpy)33+ reduction with free radical TPrA• (scheme 2).

The ECL intensity of the Ru(bpy)32+/TPrA system is influenced by the following factors: (1) Solution pH: The maximum value of the ECL intensity occurs at pH 7.5. Higher pH value (pHW9) is unfavorable due to high ECL background signal resulting from the reaction between Ru(bpy)33+ and hydroxide ions; (2) Electrode material: In aqueous solution, ECL intensity is strikingly enhanced on the glassy carbon (GC) electrode compared with that on Pt and Au electrodes, due to the increased TPrA oxidation reaction rate. Pt and Au electrodes are covered with anodic oxide layers in the ECL potential region, which inhibits the direct oxidation of TPrA. However, in the MeCN solution, the situation is different. The relative ECL intensity ratio is 100 : 93 : 61 on the Au, Pt and GC electrode; (3) Properties of the electrode surface. Rendering the electrode surface more hydrophobic can facilitate the oxidation of the TPrA and thus increase the ECL intensity. For example, modification of the Pt and Au electrodes with thiol monolayers leads to the enhanced ECL intensity due to the improved TPrA oxidation rate. Addition of surfactants (nonionic and ionic) can dramatically increase the sensitivity of this system by surface adsorption; (4) Dissolved O2. It occurs when low concentrations (<20 mM) of TPrA are used, particularly for the first ECL wave. Co-reactant oxidation generates a relatively small amount of reducing intermediates at low concentration of TPrA. O2, acted as an interceptor, destroys the intermediates before they participate in the ECL reaction;(5) Solvent media: the energy of ECL emission increases as the polarity of the solvents increases since hydrogen bonding and dipole forces can dramatically change the ground- and excited-state properties of Ru(bpy)32+. The most effective method to enhance Ru(bpy)32+ ECL (up to 270-fold in 30% 2,2,2-trifluoroethanol) is found in the mixed alcohol/water solutions compared to that in water.


Excerpted from Spectroscopic Properties of Inorganic and Organometallic Compounds by J. Yarwood, R. Douthwaite, S. B. Duckett. Copyright © 2013 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents

Electrochemiluminesence of Ruthenium Complex and its Application in Biosensors; Spectroscopic Studies of Quantum Dots; Surface Enhanced IR Spectroscopy - Principles and Applications; NQR Spectroscopy; Raman Sensors for Inorganic Salt Solutions; Spectroscopic Studies of Metal Complexes

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