**Abstract**

In electrochemiluminescence (ECL) studies, Tris (bipyridine)ruthenium(II) chloride (Ru(bpy)3 2+) and its derivatives have been used as primary luminophores since 1972. The flexible solubility in both aqueous and non-aqueous medium and the remarkable intrinsic properties like chemical, optical and desirable electrochemical behavior drives the researcher to use Ru(bpy)3 2+ and its derivatives as highly active ECL probes in modern analytical science. Novel surface modification of Ru(bpy)3 2+ based ECL platforms are highly useful in the selective and sensitive detection of biomolecules, DNA analysis, immunoassays detection, and imaging of the biologically important molecules in cells and tissue of living organisms. This chapter discusses and highlights the most significant works in Ru(bpy)3 2+ based ECL properties of reaction mechanisms and their applications.

**Keywords:** electrochemiluminescence, Ru(bpy)3 2+, biosensor, annihilation mechanism, co-reactant mechanism

## **1. Introduction**

The Electrochemiluminescence (ECL) is a process where the emission of light occurs by an excited luminophore molecule generated by reactive intermediates at the interface of the electrode and electrolyte [1]. It involves in three different kinds of processes. The first process is an electrical step, where the reactive intermediates of luminophores are generated at the electrode-electrolyte interface during the scanning of potential or applying a constant potential. In the second step, an energetic electron transfer occurs between the reactive intermediate which leads to the formation of an excited luminophore. Then the third step is a luminescence process, where the excited luminophore emits light during relaxation to the ground state. The first ECL reaction was observed by David M. Hercules in 1964, which deals with the ECL of Rubrene molecule in a non-aqueous medium [2].

#### **1.1 ECL principle**

In general, the ECL principle involves the conversion of electrical energy into radiative energy through a chemical reaction [1]. The energy required to produce an exciting luminophore molecule by the electrochemical method is referred to as a change of enthalpy which is denoted as ΔH [3]. The enthalpy change of a particular ECL reaction can be calculated by using the following equation.

$$-\Delta H \left(\text{in } eV\right) = \mathbf{E}\_0^{\text{axi}} - \mathbf{E}\_0^{\text{red}} - \mathbf{0}.\mathbf{1} \,\mathbf{6} \,\text{eV} \tag{1}$$


The energy required to generate first singlet excited state is determined by following equation.

$$\mathbf{E}\_s \left( \text{in } eV \right) = \mathbf{1} \mathbf{2} \mathbf{3} \mathbf{9}. 8/\lambda \left( nm \right) \tag{2}$$

If, -ΔH≤Es, then the ECL system is referred as energy deficiency system; for energy sufficient system, -ΔH≥ Es.

#### **1.2 Reaction mechanisms**

Normally, ECL reactions follow only two kinds of reaction mechanisms named as annihilation mechanism and co-reactant mechanism. The reaction mechanism of any ECL system depends on the reaction conditions such as the selection of luminophore, potential sweep direction, and nature of electrolyte.

#### *1.2.1 Annihilation mechanism*

In the annihilation mechanism, only luminophore molecule alone will participate in the emission of light. The luminophore get oxidizes at anode during the positive potential sweep direction (anode direction) to generate cationic intermediate or cationic radical intermediate. At the same time, the anionic radical intermediate of luminophore molecule was generated at the cathode during the potential sweep towards cathode direction. Then an energetic electron transfer occurs between highly energetic anionic and cationic reactive radicals, leads to produce one excited and one ground state luminophore molecule. The excited luminophore molecule comes to the ground state by emitting energy in the form of photons. For example, the emission of light by Ru(bpy)3 2+ molecule in acetonitrile with tetrabutylammonium tetrafluoroborate (TBABF4) as an electrolyte is the best example of annihilation mechanism [4]. The reaction mechanism is given below.

$$\left(Ru(bpy)\right)\_{3}^{2\*} \rightarrow Ru(bpy)\_{3}^{3\*} + e^{-} \left(\text{oxidation } at \text{ anode}\right) \rightarrow\_{0} = \text{1.2 V vs } \text{SCE} \tag{3}$$

$$\left(Ru(bpy)\right)\_{3}^{2\*} + e^{-} \rightarrow \left.Ru(bpy)\right|\_{3}^{1\*} \left(\text{reduction } at \text{ cathode}\right) \quad \mathbf{E}\_{0} = -\mathbf{1}.4 \text{ V } vs \text{ SCE} \tag{4}$$

$$\left(Ru(bpy)\right)\_{3}^{1+} + Ru(bpy)\_{3}^{3+} \rightarrow Ru(bpy)\_{3}^{2+} + Ru(bpy)\_{3}^{2+\*} \text{(exccitation)}\tag{5}$$

*Ruthenium-Tris-Bipyridine Derivatives as a Divine Complex for Electrochemiluminescence… DOI: http://dx.doi.org/10.5772/intechopen.96819*

$$\left(Ru(bpy)\right)\_3^{2\text{\textquotedblleft}} \rightarrow Ru(bpy)\_3^{2\text{\textquotedblleft}} + h\nu\left(\text{emission}\right) \tag{6}$$

For the sake of better understanding the above mechanism is shown in **Figure 1**. The annihilation mechanism occurs only in organic electrolytes and it requires a wider potential window in order to obtain the ECL. Because of the gas evolution reactions (oxygen and hydrogen evolution), the annihilation mechanism is not taking place in an aqueous medium.

#### *1.2.2 Co-reactant mechanism*

The drawbacks of the annihilation mechanism are overcome by adding an additional reagent called as a co-reactant along with the luminophore, and the mechanism is called a co-reactant mechanism. In this type of mechanism two reagents were taken into the account, one is luminophore and the other is a coreactant molecule. Further, the narrow potential window is sufficient to gain the ECL and it is applicable in both organic and aqueous electrolytic medium. Based on the potential sweep direction the co-reactant mechanism is further classified into two types which are discussed below.

#### *1.2.2.1 Oxidative-reduction mechanism*

The potential window is fixed to only the anodic region. The co-reactant get oxides first at the anode to form oxidizing radical intermediate which has the high

**Figure 1.**

*The schematic representation of annihilation mechanism of Ru(bpy)3 2+ molecule in acetonitrile.*

reducing ability, then luminophore oxidizes to produce cationic reactive intermediate. After that, the co-reactant intermediate reduces the luminophore intermediate to generate an excited luminophore which emits light during energetic electron transfer reaction. The ECL of Ru(bpy)3 2+ and tri-n-propyl amine (TPrA) system is the best example of an oxidative-reduction mechanism [5]. The reaction mechanism of Ru(bpy)3 2+/TPrA system is given below.

$$\left(Ru(bpy)\right)\_3^{2\*} \to Ru(bpy)\_3^{3\*} + e^- \tag{7}$$

$$\text{TPrA} \rightarrow \text{TPrA}^{\bullet \cdot} + e^- \tag{8}$$

$$\text{TPrA}^{\bullet \ast} \to \text{TPrA}^{\bullet} + \text{H}^{\ast} \tag{9}$$

$$\left(Ru(bpy)\right)\_{3}^{3+} + \text{TPrA}^{\bullet} \rightarrow Ru(bpy)\_{3}^{2+\*} + \text{pr}\_{2}\text{N}^{\*}\text{CH}=\text{CH}-\text{CH}\_{2} \tag{10}$$

$$\left(Ru(bpy)\right)\_{3}^{2\*\*} \to Ru(bpy)\_{3}^{2\*} + \mathbf{h} \tag{11}$$

Here, Ru(bpy)3 2+ is luminophore and TPrA acts as co-reactant. The schematic illustration for above reaction mechanism is also shown as **Figure 2**.

#### *1.2.2.2 Reductive-oxidation mechanism*

In this mechanism, the ECL is obtained by sweeping the potential exclusively to the cathode direction. The cathodic co-reactant gets reduced during cathodic potential scan where it produced high oxidizing ability of radical intermediate and then

**Figure 2.**

*The schematic illustration of oxidative-reduction mechanism of Ru(bpy)3 2+ molecule and TPrA.* *Ruthenium-Tris-Bipyridine Derivatives as a Divine Complex for Electrochemiluminescence… DOI: http://dx.doi.org/10.5772/intechopen.96819*

the followed by luminophores reduces to produce the anionic reactive intermediates. After that, the reduced radical intermediate of co-reactant oxidizes the anionic intermediate of the luminophore to form an excited luminophore which finally emits light. One of the classical examples for the reductive-oxidation mechanism is the ECL of Ru(bpy)3 2+/per-sulphate (S2O8 2−) [6]. The reaction mechanism of this system is shown below.

$$\mathrm{S\_2O\_8}^{2-} + e^- \rightarrow \mathrm{SO}\_4^{\bullet-} + \mathrm{SO}\_4^{2-} \tag{12}$$

$$\left(Ru(bpy)\right)\_3^{2\*} + e^- \to Ru(bpy)\_3^{1\*}\tag{13}$$

$$\left(Ru(bpy)\right)\_3^{1\*} + SO\_4^{\bullet -} \to Ru(bpy)\_3^{2\*\*} + SO\_4^{2-} \tag{14}$$

$$\left(Ru(bpy)\right)\_{3}^{2\text{\textquotedblleft}\*} \rightarrow Ru(bpy)\_{3}^{2\text{\textquotedblleft}} + h\nu\tag{15}$$

Here, Ru(bpy)3 2+ is luminophore and S2O8 2− acts as co-reactant. The above mechanism is also given as schematic diagram which is indicated as **Figure 3**.

#### **1.3 Role of Ru(bpy)3 2+and its derivatives as a luminophores in ECL**

The first ECL experiment with Ru(bpy)3 2+ as a luminophore was performed by A.J. Bard *et al.* in 1972 [4]. This discovery brought brightness to the ECL and

**Figure 3.** *The scheme of reductive-oxidation mechanism of Ru(bpy)3 2+ molecule and persulfate.*

created an endless platform for researchers to study the various kinds of ECL reactions. However, the ECL of Ru(bpy)3 2+ was limited to organic electrolytes because the ECL follows annihilation mechanism which requires wide range potential window. To overcome this problem the ECL reaction within the narrow potential window was performed by taking additional reagent along with Ru(bpy)3 2+ molecule. The first luminophore-co-reactant ECL reaction was carried out in 1981 by A.J. Bard group [7], oxalate was used as a first co-reactant to study the ECL reaction of Ru(bpy)3 2+ molecule. Later on, various types of co-reactants were discovered like tri-n-propyl amine (TPrA), triethylamine (TEA), diethylamine (DEA), NADH, ascorbic acid, 2-(dibutyl amino) ethanol (DBAE), per-sulphates, hydrogen peroxide and glutathione etc [8]. Ru(bpy)3 2+/co-reactant based ECL system plays a key role in a variety of analytical and clinical diagnostic applications. Recently *in-situ* generated co-reactants such as sulphate anion radicals and hydroxyl radicals also used as a new class of co-reactants to study the ECL of Ru(bpy)3 2+ molecule in the aqueous system by using boron-doped diamond electrode (BDD) [9]. The superior ECL luminophore activity of Ru(bpy)3 2+ molecule over the other luminophores is due to the high luminescent properties, elevated solubility in both organic and aqueous medium at room temperature, the reversible redox properties at the relevant potential region and high ECL quantum efficiency [10]. In general, Ru(bpy)3 2+ has d6 electronic configuration with octahedral structure, the emission of light is due to the metal to ligand charge transfer (MLCT) transition. The emission wavelength of Ru(bpy)3 2+ lies between 600 to 650 nm.

In addition, the derivatives of Ru(bpy)3 2+ were also shown their own contribution to ECL as luminophores. The linkage of aliphatic acids or aldehydes to the Ru(bpy)3 2+ molecule gives different kind of luminophores called as acrylates. The ECL emission wavelength of Ru(bpy)3 2+ was tuned by linking the different aliphatic compounds in acrylates (640 nm to 700 nm). And also, the Ru(bpy)3 2+ conjugated with Schiff bases shown self-enhanced ECL signal with more intense light than Ru(bpy)3 2+ molecule [11]. The enhanced ECL signal intensity is due to the resonance structure of imino radicals and presence of phenolic hydroxyl groups. Further, Ru(bpy)3 2+ and its derivatives like Ru(bpy)3 2+ dendrimers, and polypyridyl Ru-complexes used as a luminophore to study the bipolar ECL, microfluidic based ECL, wireless ECL [12, 13]. Apart from this, immobilization of Ru(bpy)3 2+ molecule on polymer-coated electrodes shown new trend and remarkable ECL behaviour and created a solid-state platform for various kinds of analytical applications. In this context, Ru(bpy)3 2+ incorporated on Nafion coated graphite electrode shows unusual ECL behaviour than solution-phase ECL system [14]. In similar way, Ru(bpy)3 2+ on Nafion coated glassy carbon electrode (GCE) shown three ECL signals in co-reactant free oxygen saturated phosphate buffer solution (PBS).

Because of the excellent ECL behaviour exhibition by Ru(bpy)3 2+ molecule, researchers tuned intrinsic properties of the Ru(bpy)3 2+ molecule by introducing different functional groups into the parent Ru(bpy)3 2+ and have been used in different analytical applications. In particular, Ru(bpy)3 2+ utilized as an ECL probe in the detection of immunoassays [15], for example, the methylcytosine which belongs to a class of immunoassay detected by using ECL sensing method [13]. The DNA detection by ECL method has carried out by using Ru(bpy)3 2+ molecule as ECL active material [16]. The double-standard DNA was detected by label-free ECL method using Ru(phen)3 2+ as an ECL luminophore [17]. Apart from this, the ECL of Ru(bpy)3 2+ also used in metal ions detection, bio-imaging, aptamer detection and other intracellular studies [13]. The schematic diagram is shown (**Figure 4**) the overall Ru(bpy)3 2+ based ECL applications.

*Ruthenium-Tris-Bipyridine Derivatives as a Divine Complex for Electrochemiluminescence… DOI: http://dx.doi.org/10.5772/intechopen.96819*

**Figure 4.**

*Schematic illustration of ECL based applications using Ru(bpy)3 2+ as a active probe.*

#### **2. Specific examples of Ru(bpy)3 2+ and its derivatives for ECL studies**

The ECL properties of various categories of Ru-based luminophores such as Ru (bpy)3 2+, Nano materials doped with Ru(bpy)3 2+ molecule, and Ru(bpy)3 2+ immobilized on Nafion coated electrode are discussed below.

#### **2.1 ECL of Ru(bpy)3 2+ complex**

The ECL of luminophore/co-reactant system follows only co-reactant mechanism either oxidative-reduction or reductive-oxidation mechanism. The initial discovery of Ru(bpy)3 2+ as luminophore shown an avenue in ECL and created a way to study the various ECL reactions. The ECL of Ru(bpy)3 2+ along with co-reactant is playing a vital role in broadening the ECL studies. Interestingly the ECL of Ru(bpy)3 2+/TPrA system follows both co-reactant and annihilation mechanism. In the case of the co-reactant mechanism TPrA gets oxidized to form TPA● and Ru(bpy)3 2+ produces Ru(bpy)3 3+ upon oxidation. The TPA● reduces the Ru(bpy)3 3+ to excited Ru(bpy)3 2+ molecule which emits light (**Figure 5A**). But in the annihilation mechanism, Ru(bpy)3 2+ electrochemically oxidizes to Ru(bpy)3 3+ and the TPA● directly reacts with Ru(bpy)3 2+ to produces Ru(bpy)3 1+, then electron transfers from Ru(bpy)3 1+ to Ru(bpy)3 3+ and generates excited Ru(bpy)3 2+ molecule which emits light (**Figure 5B**).

In the reductive-oxidation mechanism, the ECL of Ru(bpy)3 2+ occurs along with S2O8 2−, hydrogen peroxide (H2O2), and glutathione as co-reactants. The ECL of Ru(bpy)3 2+ with glutathione is quite interesting because when the reduced

glutathione (GSH) used as a co-reactant the ECL is ROS dependent. Initially, GSH reacts with reactive oxygen species (ROS) which are produced during the oxygen reduction reaction to forms GS● , then Ru(bpy)3 2+ reduces to Ru(bpy)3 1+ by electrochemically and electron transfers from Ru(bpy)3 1+ to GS● leads to the generation of excited Ru(bpy)3 2+ which emits light (**Figure 6A**). But in the case

#### **Figure 5.**

*ECL reaction mechanism of Ru(bpy)3 2+/TPrA system (A, B). Copyright © 2002, American Chemical Society.*

*Ruthenium-Tris-Bipyridine Derivatives as a Divine Complex for Electrochemiluminescence… DOI: http://dx.doi.org/10.5772/intechopen.96819*

of oxidized glutathione (GSSG), ECL is ROS independent, where the GS● forms directly by reacting with Ru(bpy)3 1+ after that excited Ru(bpy)3 2+ obtained by reacting with Ru(bpy)3 1+ and then light emission occurs (**Figure 6B**). However, the *in-situ* generated co-reactants were also utilized in order to study the ECL reaction by taking the Ru(bpy)3 2+ as a luminophore [9]. Hence, Ru(bpy)3 2+ is acting as a model and benchmark luminophore over the others. However, in order to improve further ECL light emission intensity, researchers were developed Ru(bpy)3 2+ derivatives by doping with nano-materials, the results are discussed below.

#### **2.2 ECL of Ru(bpy)3 2+-doped with nanomaterials**

Doping of nanomaterials with Ru(bpy)3 2+ leads to the newer generation of ECL luminophores. The nanoparticles with the functional groups like thiols, amines, and silicates easily covalently bind with Ru(bpy)3 2+ and its derivatives to gives highly luminescent luminophores. The Ru(bpy)3 2+-doped nanomaterials have several advantages over conventional ECL luminophores. At first, a huge number of luminophore molecules could be encapsulated in a single target molecule site secondly the self-quenching properties of luminophores will be minimized and also the external quenchers like oxygen and water molecules are screened [18]. In this sequence, Ru(bpy)3 2+ molecule were covalently linked with doped silica nanoparticles (Ru(bpy)3 2+-DSNPs) showed bright ECL signal in 0.1 M acetonitrile with Tetrabutylammonium hexafluorophosphate (MeCN/TBAPF6) potential scan from -1.6 V to +1.5 V [18]. This ECL signal is obtained by annihilation route in the absence of a co-reactant. Further, interesting ECL results were shown by making

#### **Figure 7.**

*(A, B) ECL reaction mechanism of DSNP/TPrA system. Copyright © 2009, American Chemical Society. The schematic representation of ECL generation in Ru-DSNP/TPrA system (C) Copyright © 2015, American Chemical Society.*

a self-assembled monolayer (DSNPs-SH SAM) on a gold electrode surface in the presence of TPrA [18].

There are two ECL signals were obtained at 0.91 V and 1.23 V during the potential scan from 0 to 1.6 V in 0.1 M phosphate buffer solution (PBS). For the first cycle, the ECL at 0.91 V is much intense than the second peak (at 1.23 V) [18]. In general, the TPrA oxidation on a gold electrode is prevents in PBS because of Au-oxide formation, but DSNPs-SH SAM formation on gold surface creates hydrophobic nature and suppresses the Au-oxide formation. The hydrophobic formation allows the direct oxidation of TPrA and generates more number of TPA● which directly reduces the Ru(bpy)3 2+ (in DSNPs) to form Ru(bpy)3 1+. Then Ru(bpy)3 1+ oxidized by reacting with TPrA●<sup>+</sup> to generates excited Ru(bpy)3 2+ molecule which emits light at 0.91 V (**Figure 7A**). The second peak at 1.23 V is due to electrochemical oxidation of both TPrA and Ru(bpy)3 2+ molecule (see **Figure 7B**). On the second cycle onwards the first ECL (at 0.91 V) was disappeared whereas the peak at 1.23 V remains as such because the DSNPs-SH SAM detaches from the electrode surface. This again leads to further Au-oxide growth on Au surface and suppresses the TPrA oxidation, obviously decreases ECL intensity at 0.91 V. Similarly, Ru-DSNP/TPrA exhibits ECL in PBS, the emission is due to the electron hopping between the electrode and Ru(bpy)3 2+ as well as between two adjacent Ru(bpy)3 2+ presents on DSNP as shown in **Figure 7C**. Another attempt has been carried out by covenant linkage of nitrogen-doped carbon nanodots (NCNDs) with the Ru(bpy)3 2+ molecule. The NCNDs acting as co-reactant which is electrochemically oxidized at electrode to produce reactive radicals which have the capability to form excited Ru(bpy)3 2+ molecule in generating the ECL [19]. This kind of doping of nanomaterials with the Ru(bpy)3 2+offers in enhancing the ECL intensity of Ru(bpy)3 2+ and leads to create a new platform for various analytical applications.

#### **2.3 ECL of Ru(bpy)3 2+ immobilized on Nafion coated electrodes**

The immobilization of Ru(bpy)3 2+ into the Nafion coated electrode surface either by chemical or electrochemical methods develops a highly stable and intense solid-state ECL platform. The solid-state ECL has several advantages over solutionphase ECL system. The minute amount of luminophore is sufficient to study the ECL reaction, the luminophore molecule could be regenerated on the electrode surface and also the solubility problem could be overcome. The Nafion is a cation exchange polymer with chemically inert and high thermal stability. It is used for immobilization of positively charged species like Ru(bpy)3 2+ molecules. The first reports on immobilization of Ru(bpy)3 2+ into the Nafion coated electrodes by Rubinstein and Bard have been extended to lot of studies on ECL reactions [20]. The ECL intensity of Ru(bpy)3 2+ controlled by varying the Nafion concentration, thickness of the Nafion coating on the electrode surface and amount of loading of luminophore. For example, the bright ECL observed for Ru(bpy)3 2+ immobilized on Nafion Langmuir-Schaefer coated GCE in the presence of TPrA, the highest intense ECL signal obtained for 20 layers coated electrode [21]. In a similar way, Ru(bpy)3 2+ immobilized on Nafion coated GCE electrode exhibited three ECL signals in oxygen saturated PBS [22]. The highest ECL intensity was observed when the 6.58 μm thickness of Nafion is coated on the electrode surface. In addition, the modified GCE surfaces also utilized to immobilize the Ru(bpy)3 2+ and studied the ECL reactions in order to explore a different kinds of applications [23].

However, the immobilization of Ru(bpy)3 2+ on Nafion coated noble metal surfaces like gold electrode shown superior ECL behaviour over the carbon basedelectrodes. For instance, Ru(bpy)3 2+/Nafion/gold electrode shown unique and

*Ruthenium-Tris-Bipyridine Derivatives as a Divine Complex for Electrochemiluminescence… DOI: http://dx.doi.org/10.5772/intechopen.96819*

unusual ECL properties [24]. The ECL obtained at very less cathode potentials without co-reactant in the potential region of 0 to 1.2 V in PBS. The ECL spectrum reveals that the observed ECL is because of the formation of excited Ru(bpy)3 2+ by electrochemical reaction. Similar way, the ECL of Ru(bpy)3 2+ which is presents on Nafion coated nanoporous gold electrode (NPG) is abnormal than the conventional system [25]. In this system Ru(bpy)3 2+ molecules is immobilized electrochemically in the acidic electrolyte, as prepared Ru(bpy)3 2+/Nafion/NPG composite used to study the ECL in PBS. There is a bright ECL signal obtained at less cathode potential (at 0.1 V *vs* Ag/AgCl) during the potential sweep from 0 to 1.6 V to -1 V. The *in-situ* generated hydroxyl anions (OH<sup>−</sup> ) from the NPG surface during the reduction playing a key role to gets the ECL at very less potentials. As shown in **Figure 8**, the NPG get oxidized during the potential scan from 0 to 1.6 V to form a gold-hydroxide layer (Au-OH3). At the same time, the Ru(bpy)3 2+ which is immobilized on the electrode surface (Ru(bpy)3 2+/Nafion/NPG) also undergoes oxidation to forms Ru(bpy)3 3+ intermediate. During the reduction, the Au-OH3 converts into Au0 and liberates OH− ions. The presence of Nafion does not allow diffusion of OH− ions from electrode surface to bulk electrolyte solution. It results in generation of OH● by reacting with Ru(bpy)3 3+. Then an energetic electron transfer arises from OH● to Ru(bpy)3 3+ leads to the generation of excited Ru(bpy)3 2+ molecule which emits light as indicated in **Figure 8**. This type of solid-state ECL which is obtained at very less potentials has several advantages and also has scope in various analytical application points of view. The recent trend in ECL is involving in the development of solid-state ECL for point-of-care applications. The immobilization of luminophores on polymer-coated electrodes helps in creating a solid-state platform in improving the ECL signal intensity as well as could be useful in different analytical applications.

#### **Figure 8.**

*The schematic representation of solid-sate ECL mechanism involving in Ru(bpy)3 2+/Nafion/NPG composite in 0.1 M PBS (pH 7.4).*
