Biobased, Internally pH-Sensitive Materials: Immobilized Yellow Fluorescent Protein as an Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Matrices

The pH is fundamental to biological function and its measurement therefore crucial across all biosciences. Unlike homogenous bulk solution, solids often feature internal pH gradients due to partition effects and confined biochemical reactions. Thus, a full spatiotemporal mapping for pH characterization in solid materials with biological systems embedded in them is essential. In here, therefore, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is presented. A genetically encoded ratiometric pH sensor, the enhanced superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of different materials, including natural and synthetic organic polymers as well as silica frameworks. By using controlled, tailor-made immobilization, sYFP is homogenously distributed within these materials and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. Evolution of internal pH is monitored in consequence of a proton-releasing enzymatic reaction, the hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix. Unlike optochemical pH sensors, which often interfere with biological function, labeling with sYFP enables pH sensing without altering the immobilized enzyme's properties in any of the materials used. Fast response of sYFP to pH change permits evaluation of biochemical kinetics within the solid materials. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of biology confined to the internally heterogeneous environment of solid matrices.