Acridinium Chemiluminogenic Labels—Synthesis, Analytical Performance, and Mechanism of Light Generation—A Comparison in View of Biomedical Diagnostics

This paper presents the synthesis, physicochemical characterisation, and analytical applications of chemiluminescent (CL) labels based on acridinium salts (ALs) for biomedical diagnostics. These compounds emit light as a result of oxidative reactions and represent an established class of reagents widely employed in chemiluminescence immunochemical assays (CLIAs) today. A series of structurally differentiated acridinium labels (<b>AL1</b>-<b>AL5</b>) was synthesised applying mostly original synthetic routes and purified to chromatographic purity (>90%, RP-HPLC). The compounds, including a commercial product treated as a reference, were successfully conjugated to anti-human IgG, yielding stable immunochemical reagents suitable for immunoassays with CL detection. The chemiluminescence properties of the obtained labels and their protein conjugates were investigated in aqueous buffers and in the presence of surfactants. The emission profiles exhibited characteristic flash-type kinetics with emission maxima occurring within 0.15-0.25 s after reaction initiation. The presence of surfactants more or less significantly enhanced the emission intensity, with signal increases of up to approx. 2-fold compared to surfactant-free systems. Analytical calibration demonstrated a linear response of signal derived from native labels over at least one order of magnitude of concentration, with detection limits falling in the range of 10^-9-10^-10 M, confirming the high sensitivity of the developed compounds. The experimental results were supported by theoretical studies using density functional theory (DFT), which confirmed the energetic feasibility of the CL reaction pathway and identified structural factors influencing activation barriers. Additional semiempirical calculations (PM7) indicated that the dielectric environment and proximity of ionic species can influence the reaction energetics, providing mechanistic support for the experimentally observed effects of surfactants. The results demonstrate that both molecular structure and microenvironment influence CL efficiency and kinetics of the investigated systems. The developed acridinium labels exhibit analytical performance better or comparable to commercial reagents and are fully compatible with standard immunodiagnostic conjugation protocols, confirming their suitability for use in modern chemiluminescent immunoassays.