Confocal Raman Microscopy for pH-Gradient Preconcentration and Quantitative Analyte Detection in Optically Trapped...

Area of Science:

• Analytical Chemistry
• Biophysical Chemistry
• Chemical Physics

Background:

• Vesicle membranes can maintain pH gradients, enabling isolation and preconcentration of ionizable compounds.
• Confocal Raman microscopy offers in situ observation of chemical processes within microscopic volumes.

Purpose of the Study:

• To demonstrate pH-gradient preconcentration of compounds into optically trapped vesicles.
• To develop a model predicting analyte enrichment based on acid-base equilibria.
• To assess the quantitative analysis potential for small-volume samples.

Main Methods:

• Utilized optically trapped 1 μm phospholipid vesicles as sub-femtoliter collectors.
• Employed confocal Raman microscopy for in situ observation of analyte accumulation.
• Developed a predictive model based on acid-base equilibria and used perchlorate ion as an internal standard.

Main Results:

• Achieved a concentration enrichment of (5.2 ± 0.4) × 10(5) for benzyldimethylamine (BDMA) from a 100 nM source phase.
• Demonstrated detection of BDMA from a 25 nM source phase using unenhanced Raman scattering.
• Model accurately predicted enrichment falloff due to internal buffer titration at higher concentrations.

Conclusions:

• pH-gradient preconcentration in vesicles is a viable method for analyzing ionizable compounds in small volumes.
• The predictable calibration response over four orders of magnitude supports quantitative analysis.
• Kinetics of accumulation are rapid, consistent with membrane transfer rates.