Self-Assembly of\nMicrostructured Protein Coatings\nwith Programmable Functionality for Fluorescent Biosensors

Proteins, as genetically programmable functional macromolecules,\nhold immense potential as biocompatible self-assembling building blocks,\nowing to their versatility in building coating materials and programming\ntheir functionality genetically. In this study, we demonstrate a modular\nself-assembly of protein coatings that are genetically programmable\nfor a biosensor application. We designed and produced recombinant\nfusion protein building blocks to form microstructured coatings on\ndiverse substrates, such as glass or polymers, through thermally triggered\nliquid–liquid phase separation and an orthogonal high-affinity\ncoiled-coil interaction. We incorporated fluorescence proteins into\ncoatings and controlled the protein density to enable fluorescence\nimaging and quantification in a low-resource setting. Then, we created\na coating for a calcium biosensor using a genetically engineered calcium\nindicator protein. This protein coating served as the foundation for\nour smartphone-based fluorescent biosensor, which successfully measured\nfree calcium concentrations in the millimolar range at which extracellular\ncalcium homeostasis is maintained. Using this fluorescent biosensor,\nwe were able to detect abnormal physiological conditions, such as\nmild or moderate hypercalcemia. We envision that this modular and\ngenetically programmable functional protein coating platform could\nbe extended to the development of highly accessible, low-cost fluorescent\nbiosensors for a variety of targets.