Fluorescence-based determination of cyclic adenosine monophosphate in nasal secretions of healthy and post COVID-19 patients using Tinopal CBS-X

Abstract

Olfactory dysfunction is among the most prevalent long-term complications post COVID-19 infection, largely attributed to SARS-CoV-2 induced inflammation and epithelial injury within the olfactory mucosa. Cyclic adenosine monophosphate (cAMP), a critical second messenger in olfactory transduction, may act as a biochemical indicator of impaired neuronal signaling. This study aimed to develop and validate a novel fluorescence-based method for the determination of cAMP in human nasal secretions using Tinopal CBS-X as a selective fluorescent probe. The method is based on the instantaneous formation of a 1 : 1 ion-pair complex between cAMP and Tinopal CBS-X under acidic conditions (pH 3), resulting in a pronounced bathochromic emission shift from 430 nm (blank) to 510 nm upon excitation at 348 nm. The complex exhibited a marked increase in fluorescence quantum yield (0.63 vs. 0.19 for native cAMP). The developed method presented excellent linearity (10–400 ng mL⁻¹, r² = 0.9996), accuracy (mean recovery 98.6%), precision (RSD < 2.5%), and sensitivity (LOD = 0.83 ng mL⁻¹; LLOQ = 2.52 ng mL⁻¹). Application of the human nasal samples revealed significantly lower cAMP levels in patients with post-COVID-19 olfactory dysfunction (17.69 ± 2.99 ng mL⁻¹) compared to healthy controls (69.74 ± 5.22 ng mL⁻¹, p < 0.05). The developed technique provides a low-cost alternative to previously reported LC-MS/MS, presenting sufficient sensitivity for physiological applications and demonstrating the potential of cAMP as a biomarker of post COVID-19 olfactory dysfunction.