Confocal Raman Microscopy of pH-Gradient-Based 10 000-Fold Preconcentration of Compounds within Individual, Optically Trapped Phospholipid Vesicles

Abstract

A stable pH gradient established across the membrane of phospholipid vesicle can induce the accumulation of ionizable compounds from bulk solution into the vesicle interior. This pH-gradient vesicle loading process has previously been utilized to encapsulate drugs in pharmaceutical liposomal formulations. In the present work, this process is exploited to preconcentrate dilute analytes from free solution into phospholipid vesicles, which are then detected by optically trapping individual vesicles and measuring their contents using confocal Raman microscopy. The theory of accumulation, based on the acid-base ionization equilibria of the analyte, is developed to account for depletion of the source phase and the finite buffering capacity of the vesicle interior. The model predicts that, under appropriate conditions, enrichment factors of more than 4 orders of magnitude can be realized. To test the concept, experiments were performed measuring the accumulation of benzyldimethylamine into 600-nm phospholipid vesicles. Manipulation of vesicles by optical trapping allows accumulation within an individual vesicle to be characterized while varying the external solution conditions. A more than 10 000-fold enrichment of the analyte concentration inside the vesicle relative to the source phase is reported. The results suggest that pH-gradient loading could be exploited as a powerful preconcentration scheme for trace analysis using either Raman microscopy or other microspectroscopy techniques.