Differential ATP and Pyrophosphate Sensing Using Perylene Diimide-Amino Clay Nanocomposites for Enzyme and Biofluid Assays

We report a perylene diimide (PDI)-based amphiphilic dye-Amino Clay (AC) composite that exhibits tunable photophysical and self-assembly behavior for selective phosphate sensing and enzyme detection at physiological pH in a buffered medium. Incorporating AC is crucial, as its high surface charge density, layered silicate framework, and abundant protonated amine groups enable strong electrostatic binding, aggregate modulation, and enhanced optical responses of probe molecules. Electrostatic complexation between anionic −COO– groups of the dye and NH2 sites of AC produced ∼54-fold fluorescence quenching and increased hydrodynamic diameter from 530 ± 22 to 987 ± 58 nm. ATP binding yielded a ∼42-fold fluorescence enhancement (a LOD of 10.8 ppb) via dual electrostatic and π–π stacking interactions, reducing the zeta potential to +2.8 mV. In contrast, PPi caused the displacement of dye molecules from the clay surface, inverting the zeta potential to ∼5.3 mV and reducing the particle size to 650 ± 48 nm. The system enabled the selective detection of ALP (0–30 UL–1) and the quantification of ATP in diluted serum, saliva, and urine with 36.2, 17.3, and 5.89-fold enhancements, respectively, underscoring its diagnostic potential. Such studies are vital for developing low-cost, highly selective optical sensing platforms capable of functioning in complex biological matrices for early disease diagnosis.