Tailoring cyclodextrin platforms for nanomolar range fluorescence detection of 1-hydroxypyrene as biomarker of polycyclic aromatic hydrocarbons

1-Hydroxypyrene (1HOPy) is the most widely used urinary biomarker for monitoring human exposure to polycyclic aromatic hydrocarbons (PAHs). Current analytical methods, primarily based on chromatographic separation coupled with fluorescence detection, require sophisticated instrumentation and sample pre-treatment, limiting in situ analysis. Here we investigated cyclodextrin (CD)-based supramolecular platforms as molecular recognition scaffolds for fluorescence-based detection of 1HOPy in solution. Six CD derivatives, including two monomers, hydroxypropyl-βCD (HPβCD), and γCD, two polymers, pβCD and pγCD, and two synthetic βCD dimers (βDim1, and βDim2), were systematically evaluated through UV-Vis absorption, steady-state and time-resolved fluorescence, and molecular dynamics simulations coupled with thermodynamic integration free energy calculations. Host-dependent complexation stoichiometries (1:1, 2:1, and 2:2) and binding constants ranging from 10 3 to 10 5 M⁻¹ for 1:1 complexes were obtained via multivariate global analysis. HPβCD exhibited the highest 1:1 binding affinity (K 1:1 = 1.0 × 10 5 M -1 ) with moderate fluorescence enhancement. Differently, γCD formed cooperative 2:2 assemblies displaying pyrene excimer emission at 480 nm. Most notably, a primary side βCD dimer (βDim2) promoted ground state deprotonation of 1HOPy upon complexation (K 1:1 = 2,5 × 10 4 M -1 ), generating a phenolate species with a fluorescence spectrum that is spectroscopically distinct from both free 1HOPy and other common PAH metabolites. Computational analysis, combining 100-ns molecular dynamics trajectories with thermodynamic integration binding free energies, reasonably reproduced the experimental affinity hierarchy. Preliminary tests demonstrated that 1HOPy excitation at 400 nm in the presence of 1 mM βDim2 enables selective detection of 1HOPy without interference from competing PAH metabolites (1-naphthol, 2-naphthol, 2-fluorenol, 9-fluorenol), with a linear fluorescence response over the 10-1000 nM range. These findings establish βDim2 as a promising molecular recognition element for sensitive, selective, and potentially field-deployable fluorimetric detection of 1HOPy as a PAH biomarker.