ECL reaction

A new cholesterol flow injection analysis biosensor has also been described as an application of the H2O2 ECL sensor56. In that work, the luminol electrochemiluminescence, previously studied in aqueous media, was implemented in Veronal buffer added of 0.3% triton X-100 (v/v), 0.3% PEG and 0.4% cholate to enable the solubilisation of the cholesterol and then its efficient oxidation catalyzed by the immobilized cholesterol oxidase. The ECL reaction occurred thus in a micellar medium and the performances of the H2O2 ECL sensor were investigated. 

Although the ECL phenomenon is associated with many compounds, only four major chemical systems have so far been used for analytical purposes [9, 10], i.e., (1) the ECL of polyaromatic hydrocarbons in aqueous and nonaqueous media (2) methods based on the luminol reaction in an alkaline solution where the luminol can be electrochemically produced in the presence of the other ingredients of the CL reaction (3) methods based on the ECL reactions of rutheni-um(II) tra(2,2 -bipyridinc) complex, which is used as an ECL label for other non-ECL compounds such as tertiary amines or for the quantitation of persulfates and oxalate (this is the most interesting type of chemical system of the four) and (4) systems based on analytical properties of cathodic luminescence at an oxide-coated aluminum electrode. 

Much of the study of ECL reactions has centered on two areas electron transfer reactions between certain transition metal complexes, and radical ion-annihilation reactions between polyaromatic hydrocarbons. ECL also encompasses the electrochemical generation of conventional chemiluminescence (CL) reactions, such as the electrochemical oxidation of luminol. Cathodic luminescence from oxide-covered valve metal electrodes is also termed ECL in the literature, and has found applications in analytical chemistry. Hence this type of ECL will also be covered here. 

However, ECL was not then studied in detail until 1963 [4, 5], At this time ECL from solutions of aromatic hydrocarbons was first recorded, and mechanisms involving electron transfer between electrically generated radical anions and cations were proposed. Between the mid-1960s and late 1980s there was considerable interest in the phenomenon of ECL. More than 60 publications in the literature focused almost solely on the mechanism of ECL reactions, identi-... 

The potential of ECL in analytical chemistry has only more recently been investigated, but has rapidly gained recognition as both a sensitive and selective method of detection. Most reported applications have utilized the tris(2,2 -bipyri-dyl) ruthenium(II) [Ru(bpy)32+] ECL reaction, or else the electrochemical initiation of more conventional CL reactions, but many other potentially useful systems have been investigated. The applications of ECL in analytical chemistry have recently been the subject of comprehensive reviews [12-16],... 

Most ECL reactions involve a complex series of electrochemical and CL reaction steps, such that the exact mechanism of all but the simplest ECL reactions has not been explicitly determined. This may be a problem in the search for new analytical applications, when certain compounds show marked ECL activity, while other closely related compounds are inactive, or when trying to produce a linear calibration for a particular analyte. Many applications have also been hindered by poor reproducibility in the ECL measurements, which can only be overcome by frequent refreshing of the electrode surface, the reasons for which are not well understood. 

Energetic electron transfer reactions between electrochemically generated, shortlived, radical cations and anions of polyaromatic hydrocarbons are often accompanied by the emission of light, due to the formation of excited species. Such ECL reactions are carried out in organic solvents such as dimethylformamide or acetonitrile, with typically a tetrabutylammonium salt as a supporting electrolyte. The general mechanism proposed for these reactions is as follows. 

The use of IAECL for analytical applications has now almost entirely been surpassed by techniques based on certain transition metal complexes, from which ECL reactions can occur in aqueous solution. 

Figure 2 General reaction mechanism for the ECL reaction of Ru(bpy)32+ with a tertiary amine.

Figure 3 Variation of ECL intensity with pH for the ECL reaction of tripropylamine with Ru(bpy)32+. (From Ref. 31.)...

Recently ECL reactions of Ru(bpy)32+ with other reducing agents have been documented, such as various P-diketone and some methylene compounds that have cyano and carbonyl groups [40], Hence with further research, analytical applications should arise for many classes of compounds other than amines that can act as reductants, or electrochemical precursors of reductants, capable of reacting with Ru(bpy)33+ to produce ECL. 

Figure 5 Proposed mechanism for the ECL reaction of luminol. (Reprinted from Ref. 15, with permission from, and copyright, Elsevier Science.)...

Consideration should be given to the flow rate of the sample through the detection cell. Shultz and co-workers have demonstrated the wide variability in reaction kinetics between ECL reactions, and hence the influence of flow rate on ECL intensity [60], For example, the rate constants (k) of the Ru(bpy)32+ ECL reactions of oxalate, tripropylamine, and proline were calculated to be 1.482, 0.071, and 0.011/s, respectively. Maximum ECL emission was obtained at low linear velocities for slow reactions ranging up to high linear velocities for fast reactions. That is, the flow rate and flow cell volume should be optimized such that the light-emitting species produced is still resident within the flow cell, in view of the light detector, when emission occurs. 

Hydrogen peroxide produced as a result of reactions of oxidase enzymes with analyte substrates can be sensitively determined, both directly by luminol ECL and indirectly by Ru(bpy)32+ ECL. For the latter, hydrogen peroxide is detected on the basis of its ability to diminish the ECL reaction between Ru(bpy)32+ and added oxalate, by reacting with, and depleting the concentration of, oxalate. Thus ECL intensity is inversely proportional to the concentration of analyte. This principle has been used, for example, to determine cholesterol [70],... 

Standard autoradiography film can be used to visualize ECL reactions, although Amersham does provide a number of products that have been optimized for use in this procedure. Hyperfilm -ECL and Hyperpaper -ECL are both recommended Please note that Hyperpaper-ECL has photographic emulsion on only one side, so the appropriate side must be exposed to the membrane (the emulsion side has a glossy appearance). 

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