Exploring Surface-Enhanced Raman Spectroscopy of Pyrazine-2-Carbonitrile for Indirect Label-Free Albumin Quantification in an In Vitro Endothelium Permeability Assay

Abstract

Accurate, label-free quantification of proteins, and more specifically albumin, is essential in studies aimed at monitoring transport across biological barrier tissues in vitro. Surface-enhanced Raman scattering (SERS) can deliver the sensitivity and specificity needed for such studies at physiologically relevant conditions, however, direct detection of albumin is not typically feasible at such concentrations. Here we use a small-molecule reporter (pyrazine-2-carbonitrile, PCN) that can interact both with albumin and a SERS substrate to facilitate albumin quantification. The nanoparticle surface/PCN and albumin/PCN interactions are sufficiently balanced to yield the sensitivity and specificity needed for in vitro tissue studies. The major challenge in using SERS for such assays is that the spectra of analytes can differ from their nonresonant Raman spectra, due to distinct species forming at and near the surface of the nanoparticles. Specifically, the binding of PCN to gold nanoparticles, formation of Au-PCN complexes, as well as PCN itself, contribute to the SERS spectra. We elucidate the nature of these interactions through concentration dependence studies and computational methods. Ultimately, we show that understanding these different interactions is key to quantification of albumin, at physiologically relevant albumin concentrations ranging from 0.4 to 4.4 μM using SERS spectroscopy. These data compare well with the state-of-the-art spectroscopic method, i.e., the transport of fluorescently labeled albumin across cell layers. We anticipate that this assay will stimulate analysis in in vitro models, such as organ-on-a-chip models and flow systems.