Figure 11.2. Schematics of ECL assays for measuring enzyme activity. (a) Protease activity assay, showing that ECL emission is used to follow the cleavage of the immobilized substrate, leading to the removal of ECL tag from the solid surface and hence change of ECL emission. (b) Kinase activity assay, in which antibody tagged with ECL emitter is used to specifically recognize the phosphorylated product, generated by the reaction of kinase with substrate. See insert for color representation of this figure.

probes, such as Ru(bpy)32+, offer the minimum perturbation of immune activity, protein solubility, and protein stability, because the ECL probes are small molecules (~1000 Da) and easily conjugated with the target biomolecules. (d) The ECL labels are very stable (over years), which are superior to chemiluminescence and radioisotopic labels (Bard et al., 2000; Blackburn et al., 1991). (e) ECL detection provides a larger dynamic range (six orders of magnitude) for quantification analysis. (f) ECL measurements are simple, rapid (only a few second), and easy for the automation. (g) ECL emission intensity depends on the mass and size of ECL active species, offering one of the best techniques to study the DNA intercalation (Rodriguez and Bard, 1990; Xu et al., 1994; Xu and Bard, 1995), antibody affinities, and protein-protein interactions (Xu et al., 2001). (h) The ECL instrumentation is relatively simple and inexpensive.

11.1.3. Instrumentation

ECL measurements have been carried out in conventional electrochemical apparatus with three-electrode configuration with a photodetector, such as a photomultiplier tube (PMT), a photodiode, or a charge-coupled device (CCD) camera. The annihilation ECL experiments have to be carried out in organic solvents, and its ECL emission is associated with the purity of the solvents, the supporting electrolytes, and the presence of oxygen. Thus, the b annihilation ECL experiments are performed inside an oxygen-free dry box or a sealed airtight cell (Hercules, 1964; Visco and Chandross, 1964; Santhanam and Bard, 1965; Tokel and Bard, 1972; Faulkner and Bard, 1977; Xu et al., 1996; Fan, 2004). In contrast, coreactant ECL experiments are typically performed in aqueous solutions containing the high concentration of coreactant and in the presence of oxygen, and its ECL emission has been proved to be insignificantly affected by the presence of oxygen (Bard et al., 2000; Zheng and Zu, 2005a).

The commercial ECL analyzers have achieved full automation by coupling a flow-injection system with an ECL detector, which consists of an electrochemical cell and PMT. Figure 11.3 shows the basic components of such an ECL analyzer originally developed by IGEN International. The ECL analyzer is interfaced with a computer, which controls the entire analysis process, by taking one sample at a time, measuring its ECL emission intensity, refreshing a working electrode, and cleaning up the flow-injection system. Though the ECL analyzer is designed for analysis of biomolecules immobilized on magnetic beads (surface-phase assay), it has been widely used for solution-phase and surface-phase assay and many other ECL studies. Typically, the assay is run off-line in each sample tube, which is then loaded into the instrument's carousel. Each individual sample prepared in the presence of coreactor buffer solution (e.g., TPrA) is drawn into the flow cell, and magnetically responsive beads are captured onto a working electrode (usually platinum or gold) by switching a magnetic field beneath the working electrode; the ECL emission of the sample is recorded using PMT as electrochemical potential is applied to the working electrode (Figure 11.3). Finally, the magnetic field is switched off to release the magnetic beads, and the cleanser solution is used to flush the sample and regenerate the working electrode surface electrochemically. The blank buffer is flowed through the electrochemical cell, and ECL intensity of buffer solution is measured to ensure that the flow-injection system is clean and that the electrode is ready for next measurement.

The other type of commercial ECL analyzer developed by Meso Scale Discovery measures ECL from surface-phase assays carried out directly on disposable electrodes (i.e., screen-printed carbon ink electrodes). This approach allows analysis of an array of ECL samples simultaneously and achieves the high throughput, which is essential for drug screening and genomic and proteomic analysis (Debad et al., 2004).

In addition, a variety of homemade ECL cells have been designed for different applications. For example, ECL detection systems have been developed as detectors for separation techniques, such as liquid chromatography and capillary electrophoresis, in which ECL detectors have met the demands of high speed and low volume required by separation techniques (Knight and Greenway, 1994; Danielson, 2004). Recently, a so-called "wireless ECL" detector has been introduced as a detector for analysis of three amino acids in mi-crofluid devices (Arora et al., 2001). Such an ECL detector consists of a microfabricated "U"-shape floating platinum electrode across a separation channel (Figure 11.4). The elec-trophoretic separation creates a potential between both ends of U-shape electrode, which is used to generate ECL emission. Thus, the electric field is used to drive both the separation and ECL emission, which is a very clever design and may serve as a model for the future design of ECL detector in microfluid devices.

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