Surface-Enhanced Raman Scattering pH Sensing in 3D-Printed Breast Cancer Models In Vitro.

With the increasing complexity of in vitro models for tissues, cancer, and other diseases, there is a need to develop robust biosensors capable of addressing these dynamic systems. Herein, we demonstrate the use of surface-enhanced Raman scattering (SERS)-based biosensors to monitor pH in 3D cellular models. We load breast cancer cells with pH-sensitive SERS nanotags comprising polymer-encapsulated gold nanostars functionalized with the pH-sensitive Raman reporter molecule 4-mercaptobenzoic acid, we then seed the labeled cells onto a 3D-printed hydrogel scaffold and expose the model to different stimuli: a chemotherapeutic drug, a cytotoxic surfactant, and plasmonic heating. In all cases, the SERS nanotags demonstrate pronounced responses toward an increase in pH occurring over either minutes or days (depending on the stimulus), which is associated to cell membrane damage as confirmed by cytotoxicity assays. Our sensors enable pH mapping in 3D with subcellular resolution, extending to millimeter-sized 3D regions. Machine-learning-based analysis of SERS spectra makes possible the detection of pH fluctuations following biological changes in these models. This work sets out a roadmap for applying pH-sensitive SERS nanotags in advanced 3D in vitro models or hybrid biomaterials, thereby opening new avenues for continuous monitoring of intercellular communication, proliferation, viability, and more.