**3. ECL applications**

The ECL technique has several distinct advantages over many other detection systems, such as ECL provides excellent sensitivity with a detection limit at very low concentrations (subpicomolar) because of no background signal and an extremely wide dynamic range of orders of magnitude. Also, it allows the coupling of multiple labels to oligonucleotides or peptides without affecting immune reactivity. Finally, the simple instrumentation, the highly sensitive and rapid measurement, high-throughput analysis, made ECL a powerful detecting tool in the ultrasensitive detection of biomarkers. Therefore, it is of more interest among researchers to improve sensitivity and extend the applications of ECL immunoassays, DNA sensing, Bio-imaging, and point-of-care applications. Most of the ECL analysis studies utilize Ru(bpy)3 2+ and its derivatives as ECL probe. The role of Ru(bpy)3 2+ as an ECL label in different kind of sensing applications are discussed in a detailed way as follows.

#### **3.1 Immunoassays sensing**

One of the most important analytical applications of ECL is the use of commercial bioassay based on the Ru(bpy)3 2+/TPrA systems. In which the derivatives of Ru(bpy)3 2+ are used as ECL-active labels and TPrA as an efficient co-reactant. In the area of clinical diagnosis, multi-component detection is highly adapted than single-component detection. A sandwich-type ECL immunoassay array was used for the detection of multiple protein detection by incorporating the antibody coated single-walled carbon-nanotube (SWCNT) forest micro wells surrounded by the hydrophobic polymer [26]. These carboxylated SWNTs offers a more conductive surface area for the attachment of capture antibodies for Prostate-Specific Antigen (PSA) and interleukin-6 (IL-6) at the bottom of different micro-wells by amidization. The ECL signals were measured with the CCD camera, and the limit of detection for the PSA and IL-6 was observed to be 1 pg mL−1 and 0.25 pg mL−1, which is better than the commercially available bead-based protein measurement systems. As shown in **Figure 9**, the ECL enzyme-linked immunosorbent assay was developed and used to determine the methyl-cytosine in DNA [27]. The anti-methyl cytosine antibody conjugated with acetylcholinosterase, in which the acetylcholinosterase converted acetylthiocholine (substrate) to thiocholine (product). This thiocholine

#### **Figure 9.**

*Scheme for the principle of ECL determination of methyl-cytosine. Copyright © 2012, American Chemical Society.*

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

is a bifunctional molecule that exhibits both the effect of ECL acceleration and surface accumulation *via* gold-thiol binding. Due to the accumulation and acceleration effect on ECL, the quantitative measurement of methyl-cytosine was found to have higher sensitivity with the linear range from 1 pmol to 100 pmol, which is sufficient to achieve the real DNA measurements. In order to increase the ECL emission intensity of the Ru(bpy)3 2+-derivatives based ECL system, a novel and the self-enhanced ECL luminophore of the Ru(II) complex was developed by using the poly(ethylenimine) as a co-reactant, and also to form a coil-like nanocomposite with [Ru(bpy)2(5-NH2-1,10-phen)2+] [28]. By adopting the self-enhanced ECL luminophore in the sandwich-type ECL immunoassay, ultrasensitive detection of apurinic/apyrimidinic endonuclease-1 was demonstrated for the first time with the improved detection sensitivity from pg mL−1 to fg mL−1.

The ultrasensitive detection of human C-reactive protein (CPR) was demonstrated by the addition of multiple Ru(bpy)3 2+ to a single antibody with the encapsulation of a hydrophobic compound in the polystyrene microbeads [29]. With the sandwich-type immunoassay, a high sensitive CPR detection has been achieved with the detection limit as low as 0.01 μg mL−1. The obtained LOD was found to be lower than the presently available high-sensitive CPR assay systems. Based on the previous work, a similar idea of holding multiple labels was developed by the preparation of sub-micrometer-sized liposomes containing Ru(bpy)3 2+ as the ECL-active labels for CPR immunoassay. The addition of 0.1 M TPrA in the electrolytic solution (0.1 M PBS) containing 0.1 M NaCl and 1% Triton X-100, the release of the ECL label Ru(bpy)3 2+ from the liposome can be realized. The above described approach allows the bioassay to be carried out in the aqueous solutions, which is compatible with the currently available commercial ECL instrumentation. Later, this work has been extended to the application in detecting hemagglutinin, which plays a significant role in the influenza virus infection.

#### **3.2 DNA sensors**

DNA detection is of great importance in the areas of clinical testing, forensics analyses, gene expression analysis, and biological warfare agent detections. ECL has been used as a powerful analytical tool in the DNA probe assays. Similar to the ECL immunoassays, DNA probe assays is also based on the Ru(bpy)3 2+/TPrA ECL Systems. Generally, ECL DNA probe assays can be classified into two types: (1) Label-free, and (2) Label ECL detection [13, 30]. In the year of 1991, Blackburn *et al.* first reported the use of ECL of Ru(bpy)3 2+ in developing the immunoassay and DNA probe assay for clinical diagnostics [31]. The general principle for the detection of DNA using the ECL label is outlined in **Figure 10A**. In this strategy, the ssDNA is immobilized on the electrode surface, and followed by attachment of the complementary target strand ssDNA tagged with the ECL label hybridizes with the immobilized ssDNA. Then the electrode assembly is placed in an electrolyte solution containing the co-reactant and allowed for the measurement of ECL [32]. Later, Xu *et al.* developed the ECL biosensor for the detection of DNA, based on the adsorption reaction on the film modified electrode surface. In this work, firstly tagged ssDNA was immobilized on the electrode surface coated with an organized aluminium phosphate film. By immersing the film in the DNA solution, the amount of immobilized DNA-Ru(bpy)3 2+ was determined by the ECL emission resulting from the electrochemical oxidation of Ru(bpy)3 2+ and TPrA in the solution [33]. An ultrasensitive ECL method has been developed for the detection of DNA hybridization by using polystyrene microspheres/beads, as the carriers of a huge numbers of hydrophobic ECL-active labels Ru(bpy)3[B(C6F5)4] as shown in **Figure 10B**. The label-free ECL DNA detection was achieved based on the catalytic oxidation of

#### **Figure 10.**

*Schematic diagram of solid-state ECL detection of DNA hybridization (A) Copyright © 2003, American Chemical Society. Schematic diagram of DNA hybridization on a polystyrene bead as the ECL label carrier and a magnetic bead for the separation of analyte-contained ECL label/polystyrene beads (B) Copyright © 2004, American Chemical Society.*

guanine and adenine base using a glassy carbon electrode modified CNT/Nafion/ Ru(bpy)3 2+ has been reported [34]. The ECL signals for the dsDNA and their denatured counterparts could be detected with a very lower concentration of 30.4 nM and a single-base mismatch gene p53 gene sequence segment was detected with the concentration of 0.4 nM.

#### **3.3 Bio-imaging**

An important breakthrough in the analytical application of the ECL technique is the combination of this transduction technique with microscopy. The combination of ECL along with a microscope leads to marvelous investigation in imaging of cell and tissue of living beings, nanomaterial imaging, and imaging of single-particle collisions [35]. The ECL imaging setup consists of (i) bright-field microscopy, (ii) a potentiostat, and (iii) the charge-coupled device (CCD) for recording the image [36]. The ECL emission signal is directly proportional to the concentration of the luminophore and the co-reactant; hence, the ECL technique enables the sensitive detection and quantification of both luminophore and the co-reactant. This widens the application of the ECL technique in detecting various biomolecules, such as proteins, enzymatic substrates, and nucleic acids.

In the most of Bio-imaging analysis, the Ru(bpy)3 2+ molecule utilized as the ECL luminophore due to high quantum efficiency. For example, Valenti *et al.* used Ru(bpy)3 2+ as ECL label in imaging the single Chinese Hamster Ovary (CHO) cell [37]. Initially, the cells are cultured on a glassy carbon electrode and incubated with biotin X which is capable of reacting with amino groups of the protein. Then the

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

Ru(bpy)3 2+ molecule is labeled with streptavidin (SA@Ru). The biotin group reacts with SA@Ru and attaches the cell membrane throughout the ECL measurements as shown in **Figure 11a**. Further similar strategy used to imaging the MCF10A cell, in this case, CNT electrode used to incubate the cells and Ru(bpy)3 2+ labeled with monolocal antibody (Ab@Ru) serves as ECL probe as shown in **Figure 11b**. The ECL imaging has performed in the PBS (pH 7.4) containing TPrA at anodic applied potential of 1.35 V. **Figure 11c** and **d** displays the PL image and ECL image of CHO cells on GC electrode. PL image shows a spatial distribution of SA@Ru labels with an entire cell appearance (**Figure 11c**). But in the case of the ECL image, the cell border glows with red color whereas cell nucleus is dark (**Figure 11d**). This is because ECL labels (SA@Ru) specifically located on the target cell. Thus the ECL is more specific to image single cell over the PL. Similar kind of results was observed in the case of MCF10A cell, the PL shows the emission of the light entire the cell (**Figure 11e**) whereas ECL image emits light only at a specified cell membrane (**Figure 11f**). Apart from this, the microelectrode arrays also used in single cell imaging by adopting ECL strategy [38].
