Fluorescence-Based Real-Time Observation of Early-Stage Nucleation of N-Phenylanthranilic Acid

Direct observation of prenucleation processes remains challenging due to the small size and dynamic nature of early aggregates. Here, we develop an in situ spectroscopic method based on the aggregation-induced emission (AIE) of N-phenylanthranilic acid (NPAA) to probe early-stage aggregation in solution. The fluorescence response enables the identification of a prenucleation aggregate (PNA) state prior to the onset of macroscopic turbidity, allowing real-time tracking of the structural evolution from dispersed molecules to crystalline phases. Pathway-controlled antisolvent addition experiments suggest that the onset of PNA formation correlates with the overall solution composition rather than the specific kinetics of antisolvent addition, while the subsequent evolution shows dependence on the pathway of solvent composition change. Molecular dynamics simulations further support the formation of PNA by revealing finite-sized aggregates with evolving intermolecular interactions and structural organization. These results establish a direct link between AIE response and prenucleation aggregation and support a two-step nucleation pathway involving an intermediate state, providing a spectroscopic route for investigating early-stage nucleation processes.