Preconcentration of biological thiols from large volume of exhaled breath condensate using microcolumns with immobilized gold nanoparticles

A novel microcolumn-based preconcentration strategy for the selective extraction of biological thiols from exhaled breath condensate (EBC) was developed using gold nanoparticles (AuNPs) immobilized on strong anion exchange (SAX) solid-phase extraction material. The approach combines the high affinity of thiol compounds toward gold surfaces with the robustness and compatibility required for flow-based solid-phase extraction. Citrate-stabilized AuNPs were immobilized onto commercially available SAX particles via electrostatic interactions and packed into disposable microcolumns. Biological thiols, including homocysteine, cysteine, and glutathione, were selectively retained from 1–10 mL EBC samples, released using dithiothreitol, derivatized with naphthalene-2,3-dicarboxaldehyde/cyanide, and analyzed by capillary electrophoresis with laser-induced fluorescence detection (CE–LIF). The immobilized AuNP approach eliminated centrifugation steps required in conventional colloidal nanoparticle protocols and enabled rapid, reproducible, and easy sample preparation. Under optimized conditions, preconcentration factors of 57.3, 44.0, and 8.8 were obtained for homocysteine, cysteine, and glutathione, respectively. Limits of detection reached 0.05 nM for homocysteine, 0.04 nM for cysteine, and 0.58 nM for glutathione. Relative standard deviations of migration times were below 1.8%, while peak area RSDs remained below 18%. The method was successfully applied to native EBC samples, where endogenous thiols were detected at low nanomolar and subnanomolar concentration levels. The developed AuNP–SAX microcolumn system represents a simple and highly sensitive platform for ultra-trace thiol analysis in non-invasive biological matrices with strong potential for routine biomedical applications.