| Summary: | We report a fluorescent assay for the determination of vitamin B<sub>12</sub> (VB<sub>12</sub>) based on the inner filter effect (IFE) of 1,3-propanedithiol-functionalized silver nanoparticles (PDT-AgNPs). PDT was simply functionalized on the surface of AgNPs through Ag–thiol interaction, which leads to significantly enhanced fluorescence, with excitation and emission at 360 and 410 nm, respectively, via their thiol-mediated aggregation. Since target VB<sub>12</sub> has strong absorption centered at 360 nm, which is almost completely overlapping with the excitation spectra of PDT-AgNPs, the VB<sub>12</sub> induced strong quenching of the fluorescence of PDT-AgNPs via IFE. The IFE-based mechanism for the fluorescence quenching of PDT-AgNPs in the presence of VB<sub>12</sub> was confirmed by the analyses of Stern–Volmer plots at different temperatures and fluorescence decay curves. The fluorescence-quenching efficiency of PDT-AgNPs was linearly proportional to the concentration of VB<sub>12</sub> in a wide range of 1 to 50 μM, with a lower detection limit of 0.5 μM, while preserving excellent selectivity toward target VB<sub>12</sub> among possible interfering molecules. Furthermore, the PDT-AgNPs-mediated assay succeeded in quantitatively detecting VB<sub>12</sub> in drug tablets, indicating that PDT-AgNPs can serve as an IFE-based fluorescent probe in pharmaceutical preparations by taking advantages of its ease of use, rapidity, and affordability.
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