Self-Assembly of Microstructured Protein Coatings with Programmable Functionality for Fluorescent Biosensors

Abstract

Proteins, as genetically programmable functional macromolecules, hold immense potential as biocompatible self-assembling building blocks, owing to their versatility in building coating materials and programming their functionality genetically. In this study, we demonstrate a modular self-assembly of protein coatings that are genetically programmable for a biosensor application. We designed and produced recombinant fusion protein building blocks to form microstructured coatings on diverse substrates, such as glass or polymers, through thermally triggered liquid-liquid phase separation and an orthogonal high-affinity coiled-coil interaction. We incorporated fluorescence proteins into coatings and controlled the protein density to enable fluorescence imaging and quantification in a low-resource setting. Then, we created a coating for a calcium biosensor using a genetically engineered calcium indicator protein. This protein coating served as the foundation for our smartphone-based fluorescent biosensor, which successfully measured free calcium concentrations in the millimolar range at which extracellular calcium homeostasis is maintained. Using this fluorescent biosensor, we were able to detect abnormal physiological conditions, such as mild or moderate hypercalcemia. We envision that this modular and genetically programmable functional protein coating platform could be extended to the development of highly accessible, low-cost fluorescent biosensors for a variety of targets.

Keywords: biosensors; calcium indicators; coiled coils; elastin-like polypeptide; hypercalcemia; protein coating; protein engineering; self-assembly.