Electrochemiluminescence Method

Electrochemiluminescence combines electrochemical reactions and luminescence, converting electrical energy to light. Electrochemiluminescence differs from chemiluminescence; in electrochemiluminescence, the reactive species that produce the chemiluminescent reaction are electrochemically generated from stable precursors at the surface of an electrode. Luminophores are substances that emit light. In electrochemiluminescence, luminophores attain a high-energy state induced by electron transfer at the electrode surface through an oxidation-reduction reaction. The excited luminophores emit light as photons while returning to the ground state. Luminophores can be used as labels for biomolecules; the biomolecules can be detected and quantified by measuring the amount of light emitted. Electrochemiluminescence is an important diagnostic technique known for its versatility and numerous advantages. The applications of electrochemiluminescence include detecting, separating, and quantifying various intracellular and extracellular biomolecules, including proteins, enzymes, hormones, metabolites, and nucleic acids. Electrochemiluminescence is also used to visualize cells, study the functions of various intramembrane and transmembrane proteins, detect nucleic acids of interest, and assay drugs. Electrochemiluminescence systems are classified into 2 types—ion annihilation or co-reactant systems. In ion annihilation systems, a pulsed potential applied to the electrode generates radical cations and anions of the luminophore. The electron transfer between anions and cations results in an excited cation. The subsequent decay of this excited cation to the ground state results in the emission of light. Ion annihilation systems typically use organic compounds dissolved in organic solvents, donor-acceptor conjugated molecules. These organic compounds are typically poor candidates for biomolecular assay labels. Ion annihilation also generates highly reactive intermediates unsuitable for routine assays. The majority of electrochemiluminescence systems currently in use are co-reactant systems. These systems use a high-efficiency co-reactant added to the luminophore with one-directional potential scanning. Oxidation or reduction of both species at the electrode generates radicals. Intermediates from the co-reactant decompose, forming a robust species that reacts with the luminophore, producing excited states and emitting light. Co-reactant systems are used for biomolecular assays due to the solubility of the co-reactant in the surrounding medium, low reduction-oxidation potential, and stability. Ruthenium metal ions and luminol derivatives are the most commonly used lumiphores in co-reactant electrochemiluminescence systems. The co-reactant commonly used with ruthenium metal ions is tripropylamine. Other widely used co-reactants include 2-(dibutylamino)ethanol, peroxydisulfate, and hydrogen peroxide. Most reported electrochemiluminescence applications for immunoassay or genetic analysis use tris(2,2′-bipyridyl)ruthenium as a label and tripropylamine as a co-reactant. These systems are highly efficient, as the ruthenium compound is stable, highly soluble in polar and nonpolar solvents, and exhibits strong luminescence. Tripropylamine undergoes oxidation with potential application, forming a tripropylamine radical cation and a tripropylamine radical. These radicals generate excited bipyridyl-ruthenium, which emits orange-spectrum light at 600 to 640 nm as it relaxes to the ground state. The luminophore is regenerated after emission.  Luminol is an organic luminophore commonly used for cell imaging. Upon oxidation, it forms a diazaquinone intermediate, which further oxidizes to 3-aminophthalate in the presence of hydrogen peroxide, emitting blue light. Hydrogen peroxide, generated in biological processes, is often detected alongside luminol. Reactive oxygen species can enhance luminol electrochemiluminescence emission. Luminol is irreversibly oxidized and requires alkaline conditions, limiting cellular analysis applications. However, it operates at a lower anodic potential compared to bipyridyl-ruthenium, providing advantages for imaging living cells.  The basic instrumentation setup in an electrochemiluminescence includes an electrochemical cell, a detector, a signal amplification system, and a reagent and sample delivery system. The electrochemical cell houses the working electrode, typically made of carbon or gold, which serves as a site of the electrochemiluminescence reaction. A reference electrode is also present to maintain a stable potential for measurement. A photomultiplier tube or a photodiode is commonly used to detect the light emitted during the electrochemiluminescence reaction. These detectors are highly sensitive to low light levels and convert the photons into electrical signals. The electrical signals generated by the light detection system are weak and must be amplified and processed for accurate measurement. Amplification circuits, such as transimpedance amplifiers, can boost signal strength, and signal processing units can filter and digitize the signal for analysis. In an electrochemiluminescence assay, reagents and samples are delivered to the electrochemical cell using a syringe pump or a microfluidic system, which precisely administers the required volumes at specific time points. Electrochemiluminescence systems have several advantages.The luminophores used in electrochemiluminescence are small, stable substances that can label a wide range of molecules and haptens without cross-reaction. There is minimal background interference in electrochemiluminescence because the luminophore has the inherent capacity to emit light, and no additional light source is required. The technique is susceptible due to multiple excitation cycles and enables detection at very low limits, as low as 200 fmol/L. In addition, electrochemiluminescence provides improved reagent stability. Electrochemiluminescence is susceptible to light leaks and background luminescence from reagents. The high sensitivity offered by electrochemiluminescence requires pure reagents and solvents. In addition, high-intensity light emission may lead to pulse pile-up, resulting in underestimating light emission.