**3.4 Aptamer-based sensors**

Aptamers are peptide or oligonucleotide molecules and also a functional DNA or RNA structures. Aptamers can specifically bind with small molecules as well as peptides and proteins. Aptamers exhibit remarkable advantages over conventional molecular recognition systems like antigen-antibody interactions because of their easier synthesis protocols, simple labeling, and high stability [30]. The combination ECL tool along with aptamer showed attractive results in biosensor applications and several aptamer-based ECL biosensors have been developing to detect small

#### **Figure 11.**

*The schematic illustration of principle involving in ECL imaging (a, b) PL and ECL images of SA@Ru attached CHO cells (c, d) and PL, ECL images of Ab@Ru attached MCF10A cells (e, f) Copyright © 2017, American Chemical Society.*

molecules. In most of the aptamer-based ECL sensors, the Ru(bpy)3 2+ molecule and its derivatives using as ECL label along with TPrA as co-reactant, due to unique and high quantum efficiency. For example, thrombin was detected by Ru(bpy)3 2+ molecule as ECL probe, in this the captured aptamers with AuNPs labels were immobilized on thiolated ITO electrode via Au-S linkage and then catches the target aptamer [39]. After that, the Ru(bpy)3 2+ molecule label was tagged with aptamer and studied the ECL experiments in TPrA containing PBS. This ECL strategy allows the detection of thrombin at a very low level with 10 nM of LOD. In addition to this, thrombin is detected by using tris(1, 10-phenanthroline) ruthenium ion (Ru(phen)3 2+) intercalated into double standard DNA (dsDNA) as ECL sensing probe along with antithrombinthiolated aptamer [40].

The modification of gold electrode for ECL sensor is shown in **Figure 12**. Initially, the gold electrode soaked in a solution containing 2-mercaptoethanol in order to block the electrode exposing surface, then antithrombin aptamer adsorbed on the electrode surface as shown in **Figure 12A**. After that ds-DNA is placed between the aptamer and ss-DNA to intercalate the luminophore molecule (**Figure 12B**). To intercalate the Ru(phen)3 2+ into the ds-DNA, the modified electrode is dipped in the ds-DNA solution. ECL experiments performed in PBS (pH 7.5) containing TPrA, a sharp ECL signal observed at 1.1 V which is due to the energetic electron transfer between reactive intermediates of Ru(phen)3 2+ and TPrA. The ECL intensity decreases during the addition of 5 pM of thrombin into the electrolytic solution [40]. The decrease in the ECL signal is due to the detachment of Ru(phen)3 2+ molecule from ds-DNA and occupation by thrombin as indicated in **Figure 12D**. This ECL methodology provides to sense the thrombin in the range of 0.05 to 50 pM with the detection limit of 0.02 pM.

#### **Figure 12.**

*Schematic representation of the principle involving in thrombin detection by aptamer-based ECL sensor. (A) The adsorption of thiolatedantithrombin aptamer on and the 2-mercaptoethanol block to the electrode. (B) The formation of the ds-DNA between aptamer and its complementary ss-DNA. (C) The intercalation of Ru(phen)3 2+ into the ds-DNA sequence. (D) Dissociation of ds-DNA and release of Ru(phen)3 2+ due to the interaction between thrombin and its aptamer, resulting in the decreased ECL emission which was used to quantify thrombin. Copyright © 2009, American Chemical Society.*

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

### **3.5 Metal ion detection**

Heavy metal ions such as Hg, Pb, Sn and, Cd are highly toxic and undegradable, causes serious health issues to humans [41]. The heavy metal ions migrate into the soil from industrial wastage, pesticides, fertilizers, spilling of petrochemicals. As migrated metal ions contaminants food and food security has become a worldwide worry. For the past few decades, the ECL has been using as one of the leading analytical technique in detecting such metal pollutants in food as well as soil. In this context, bipolar ECL (BP-ECL) has employed in detecting the Cu2+ and Cd2+ ions [42]. This method offers high sensitivity, low cost and provides both qualitative and quantitative information. The mixed luminophores of Ru(bpy)3 2+ molecule along with Ir(ppy3) acting as ECL sensing probe. The multicoloured ECL emission is obtained at 540 nm (green) 610 nm (orange) at the driving potential of 5.5 V in the presence of both luminophores along with TPrA. The BP-ECL method offers the determination of Cd2+ ion in the range of 1 μM to 75 μM with the limit of detection of 0.094 μM (10 ppm) and also the Cu2+ ion detected within the dynamic range of 0.1 μM to 1/75 μM with 0.008 μM of LOD. In addition to this, Ru(bpy)3 2+ linked with crown ethers used as an ECL probe to determine the Pb (II) ion [43], and also the solid-state ECL has been employed by using Ru(bpy)3 2+ as an ECL probe to detect the Pb (II) ion in trace level [44]. The Pb(II) ion detected in the linear range of 1x10−6 μM to 1x102 μM by ECL turn-off method. Another class of heavy metal Hg (II) ion detected by the employing cathodic ECL of Ru(bpy)3 2+/NHS system [45]. This system includes the zero background signal, wide dynamic detection range and highly sensitive and selective to detect Hg (II) in physiological pH. The ECL intensity gradually increases upon each addition of Hg (II) ion into the solution in the linear range of 0.001 μM to 20 μM. This cathodic ECL method shows the limit of detection of 0.1 nM towards Hg (II) detection without interfering with other metal ions.

## **3.6 Point-of-care applications**

Now a day's ECL becomes one of the dynamic, highly sensitive, and well established tools in point-of-care applications. ECL strategy has been used in the fabrication of point-of-care testing devices (POCT) like ECL detectors,

#### **Figure 13.**

*Schematic diagram of a paper-based microfluidic ECL sensor. Using inkjet-printed paper fluidic substrates and screen-printed electrodes, this ECL sensor can be read with mobile phone cameras. Copyright © 2011, American Chemical Society.*

bipolar ECL devices, wireless ECL, and microfluidic chips. These POCT devices are designed by using luminophores as sensing probes, the Ru(bpy)3 2+ is a benchmark material among the various luminophores due to high quantum efficiency. The portable devices with lower prices could be useful for hospital/ nonhospital purposes. The paper-based ECL sensors are one of the best cheapest ECL devices to detect biological molecules at trace level [46]. This type of ECL sensor has advantages that possess high sensitivity, selectivity, and rapid detection. The paper-based microfluidic devices made by patterning the papers into hydrophilic channels separated by hydrophobic barriers which allow the uniform distribution of samples into the regions. The photoresist materials like wax, polydimethylsiloxane, alkylketene dimer, polystyrene, poly(o-nitrobenzyl methacrylate), fluorochemicals, methylsilsesquioxane, and toner used as hydrophobic barriers [47]. Yan *et al.* developed a paper-based ECL 3D device. The device made with two different kinds of patterned cellulose papers called paper-A and paper-B. These two papers were converted into screen printed working paper electrode (SPWPE) by using carbon paste, the Ag/AgCl used as a reference electrode. As fabricated SPWPE used for point-of-care applications. The Ru(bpy)3 2+ used as an ECL sensing probe along with TPrA as a co-reactant. The ECL signal intensity enhanced by 10-fold in the presence of CEA antibody [46]. Apart from this, microfluidic-based ECL sensor shown much attention in pointof-care applications due to flexibility, cheaper cost, portability, and porosity. **Figure 13** represents the schematic illustration of paper-based the microfluidic ECL sensor device. The inkjet printing is used to prepare microfluidic substrate, then it is combined with screen-printed electrodes which produce portable, disposable and photo-detectorless ECL sensor device. Initially, the printed paper based microfluidic filled with 13 μl of 10 mM Ru(bpy)3 2+ solution and allowed to dry. After drying the paper microfluidic substrate is further laminated on Zensor screen-printed electrode (SPE) with 80 μm thickness. The small slit is made during the lamination to introduce the sample. The Ru(bpy)3 2+ serves as ECL sensor probe and the mobile phone used to detect the ECL signal. The conventional photodetector used to detect the DBAE and NAD in the range of 0.9 μM and 72 μM respectively. In addition to this, the wireless ECL and bipolar ECL sensors also used as in fabricating the point-of- care testing devices for hospital/ nonhospital purposes.
