A novel urease-assisted ratiometric fluorescence sensing platform based on pH-modulated copper-quenched near-infrared carbon dots and methyl red-quenched red carbon dots for selective urea monitoring

A novel and sensitive fluorescence ratiometric method is developed for urea detection based  on the pH-sensitive response of two fluorescent carbon dot (CD) systems: R-CDs/methyl red (MR) and NIR-CDs/Cu²⁺. The sensing mechanism involves breaking down urea using the enzyme urease, releasing ammonia and increasing pH. At higher pH, the fluorescence of NIR-CDs is quenched due to the enhanced interaction with Cu²⁺, while the fluorescence of R-CDs is restored as the acidic MR converts to its basic form, removing the inner filter effect. The ratiometric signal (F₆₀₈/F₇₅₀) of the R-CDs/MR and NIR-CDs/Cu²⁺ intensities changed in response to the pH induced by urea hydrolysis, enabling selective and sensitive urea detection. Detailed spectroscopic and morphological investigations confirmed the fluorescence probe design and elucidated the sensing mechanism. The method exhibited excellent sensitivity (0.00028 mM LOD) and linearity range (0.001 – 8.0 mM) for urea detection, with successful application in milk samples for monitoring adulteration, demonstrating negligible interference and high recovery levels (96.5% to 101.0%). This ratiometric fluorescence approach offers a robust strategy for selective urea sensing in complicated matrices.