Localized entrapment of green fluorescent protein within nanostructured polymer films

Abstract

Protein entrapment within ultrathin polymer matrices is of significant interest for applications in biosensing, drug delivery, and bioconversion; however, it remains a big challenge due to insufficient control over protein distribution inside the matrices. We report on nanostructured protein-polyelectrolyte (PE) films obtained through localized incorporation of green fluorescent protein (GFP) within poly(styrene sulfonate)/poly(allylamine hydrochloride) matrices assembled via the spin-assisted layer-by-layer method. Film compositional and structural analyses were performed by using a combination of techniques such as neutron and X-ray reflectometry, circular dichroism (CD), atomic force microscopy (AFM), spectroscopic ellipsometry, and Attenuated Total Reflection Fourier transform infrared spectroscopy (ATR-FTIR). By using deuterated GFP as a marker for neutron scattering contrast we have inferred the architecture of the films both in vertical and lateral directions. We found that films assembled with a single GFP layer confined at various distances from the substrate exhibit a strong localization of the GFP layer without intermixing into the PE matrix. The GFP volume fraction approached the maximum value of a monolayer packing density of randomly oriented GFP molecules. However, partial intermixing of the GFP with polymeric material is evidenced in multiple-GFP layer films which showed alternating protein-rich and protein-deficient regions. Our results yield new insights into the organization of immobilized proteins within polyelectrolyte matrices and open opportunities for fabrication of protein-containing films with well-organized structure and controllable function, a crucial requirement for advanced sensing applications.