Analysis of molecular populations at liquid/solid interfaces is critical to the investigation, understanding, and optimization of interfacial chemical reactions (surface modification, coupling agents, immobilized reagents or ligands), chemical separations, and surface-based biosensing. Quantifying interfacial molecular populations is challenging, however, because of the limited number of molecules that reside at a liquid/solid interface, the difficulty of preparing interfacial standards of known surface coverage to calibrate a measured response, and interferences from bulk solution molecules. These challenges can be addressed through the use of total-internal-reflection fluorescence microscopy to quantify interfacial populations by counting individual molecules. This measurement can also report reversible reaction kinetics at equilibrium by recording ‘movies’ of individual molecular binding events. We have applied this methodology to study the kinetics of DNA hybridization and to investigate hybridization of individual immobilized DNA probe molecules. Despite the outstanding sensitivity of single-molecule fluorescence imaging, this technique requires that target molecules be fluorescently labeled. Labeling requires an additional step in an analysis, and furthermore, the label can interfere with the reaction being investigated. A label-free approach to investigate molecular interactions at liquid/solid interfaces is based on confocal Raman microscopy, where small Raman scattering cross sections can be overcome by interrogating interfacial chemistry within very high surface-area porous supports. The internal surfaces of porous silica particles can be modified with ligands and used as substrates for investigating molecular-recognition assays inside of individual particles. With these substrates, calibrated with scattering from internal standards, confocal-Raman microscopy can provide structural information and absolute surface coverages of unlabeled molecules involved in interfacial molecular-recognition reactions.

Spectroscopy through the microscope: Chemical analysis at liquid/solid interfaces

2019 Fall National Meeting

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