In Vivo Real-Time Detection of Phenylephrine in Mice Using Boron-Doped Diamond Microelectrodes with Simultaneous Video-Based Assessment of Pupil Size.

Precise quantification of ocular pharmacokinetics remains a critical bottleneck in ophthalmic drug development, primarily constrained by the inability to monitor intraocular concentrations in real time. Current methods typically rely on destructive sampling, which precludes longitudinal assessment in individual subjects. Here, we report an electrochemical platform for time-resolved quantification of phenylephrine in the anterior chamber of living mice using needle-shaped boron-doped diamond microelectrodes. An optimized multistep potential protocol mitigated biofouling and enabled stable monitoring of intraocular drug dynamics while simultaneously assessing pupillary response via video. Across a range of topical doses, intraocular exposure increased with applied concentration, whereas pupil dilation showed a saturating dependence and clear pharmacokinetic-pharmacodynamic (PK/PD) hysteresis, consistent with a temporal lag between local drug availability and physiological response. This platform enables time-resolved in vivo PK/PD analysis and provides a practical framework for evaluating and optimizing ocular drug delivery strategies.